Substituted bis-indole derivatives useful as contrast agents, pharmaceutical compositions containing them and intermediates for producing them

ABSTRACT

Metal-complexable substituted bis-indole derivatives comprising the structure shown in formula (I) hereunder: enantiomers and pharmaceutically acceptable salts thereof and metal complexes thereof, wherein L, R 1 , R 2 , R 3 , C 1 , C 2 , m, n, p, q and r are as defined in claim  1  for use as constrats agents.

The present invention relates to a novel class of bis-indole derivativesuseful as tools in biochemical, biomedical and medical applications. Inparticular, the present invention relates to the use of these novelcompounds or pharmaceutically acceptable salts and formulations thereofin therapeutic and/or diagnostic applications, in particular as contrastagents for the identification and visualization of tissues and organs.More particularly, certain such compounds are useful for theidentification and visualization of necrosis and necrosis relateddiseases including myocardial and cerebral infarction. The presentinvention also relates to methods for preparing such bis-indolederivatives, as well as intermediates therefore.

BACKGROUND OF THE INVENTION

A few disubstituted or trisubstituted indole and bis-indole derivativesare already known in the art as having therapeutic activity, as shown bythe following brief review. 3,5-disubstituted indoles such as serotonineand melatonine are well known. 2,3-disubstituted indoles wherein the 2-and 3-substituents together form a ring next to the pyrrole ring areknown e.g. from U.S. Pat. No. 4,430,269, U.S. Pat. No. 5,811,551 andU.S. Pat. No. 6,147,076. U.S. Pat. No. 5,808,064 further discloses2,3-disubsbtuted indoles wherein the 2-substituent is a trialkylsilyl ortriphenylsilyl group. U.S. Pat. No. 5,877,329 discloses2,3,5-trisubstituted indoles being useful intermediates in preparingbiologically active compounds such as certain lipoxygenase inhibitors.U.S. Pat. No. 5,932,743 discloses 2,3-disubstituted indole-6-carboxylicacids having estrogen agonist activity. U.S. Pat. No. 5,573,999discloses that 2,4-disubstituted indoles can be prepared from2-haloanilines and enamines in the presence of a Pd compound (J.Heterocycl. Chem. (1987) 24:1555). U.S. Pat. No. 3,954,799 disclosesthat synthesis of 1,3-disubstituted indoles generally requires thevigorous conditions used by Norland et al., J. Am. Chem. Soc. (1960)82:5143 for the condensation of 2-carboxybenzaldehyde and indoles. U.S.Pat. No. 6,245,761 discloses alkylation of 1,2-disubstituted indoles byway of a Mannich reaction.

With respect to bis-indoles, however, a few disubstituted bis-indolesare already known in the art. A first class of 2,2′-dicarboxylic acid(ester)-5,5′-diindolyl derivatives wherein the indole groups are linkedthrough a single bond, oxygen, sulfur, methylene or ethylene, all beingmade with yields ranging from 46 to 67% by the cyclization ofdihydrazones, are known from the publications of Samsonyia et al. inChem. Heterocycl. Compds (1981) 1:57–61 and Chem. Heterocycl. Compds(1982) 3:348–351. Additionally, Chemical Abstracts (1961) 55:5457discloses making 2,2′-dicarboxylic acid (ester)-3,3′-diindolyl sulfidewith a 40% yield by reacting SOCl₂ with indole-2-carboxylate. Chem.Heterocycl. Compds (1978) 2:217–224 discloses making2,2′-dicarbethoxy-3,5′-diindolyl with a yield of 35% by the cyclizationof a hydrazone.

EP-A-887.348 additionally discloses substituted [bis-indol-3-yl)methyl]benzenes wherein the phenyl group of the indole linking bridge issubstituted with hydroxy or carboxy, and wherein the phenyl rings of theindole groups are symmetrically substituted in the 5-position or6-position with hydroxy, bromo, amino, methylamino, diethylamino,isopropyl or ethylthio, these compounds being useful as antitumor andantimetastatic agents. The same document also discloses another type ofdisubstituted bis-indole, namely2,3-dihydroxy-1-[bis(2-hydroxyindol-3-yl)methyl] benzene. Thesecompounds can be prepared by condensing benzaldehyde with at least twoequivalents of a substituted indole.

International patent application published as WO 98/50357 discloses agroup of 5,5′-disubstituted- or 2,2′-disubstituted- orN,N′-disubstituted-3,3′-diindolylmethanes being useful as antiestrogens.In particular there are disclosed2,2′-di(C₁–C₅)alkyl-3,3′-diindolylmethanes,1,1′-di(C₁–C₅)alkyl-3,3′-diindolyl-methanes,5,5′-dihalo-3,3′-diindolylmethanes,5,5′-di(C₁–C₅)alkyl-3,3′-diindolyl-methanes and5,5′-di(C₁–C₅)alkoxy-3,3′-diindolylmethanes. They can be obtained bycondensing formaldehyde with commercially available substituted indoles,although this has the disadvantage of forming by-products whichcomplicate purification of the desired product. Therefore a preferredsynthesis in three steps consists of first forming a substitutedindole-3-aldehyde, then reducing it into the corresponding substitutedindole-3-methanol which is then condensed for example by treatment witha phosphate buffer having a pH around 5.5.

International patent application published as WO 00/09169 discloses aclass of labeled non-porphyrin compounds being e.g. applicable asdiagnostic agents in Magnetic Resonance Imaging applications or nuclearmedicine, these compounds comprising (i) an agent suitable for targetinga specific organ and/or tissue and comprising one or more organic ringcompounds, (ii) an agent suitable for labeling the targeted organ and/ortissue, and optionally (iii) a spacing agent arranged between thetargeting agent and the labeling agent. Primarily described in thelatter document are pamoic acid derivatives, i.e. compounds wherein thelabeling agent and optionally the spacing agent are in β position withrespect to the linking agent. Some of these compounds, such as thegadolinium complex of a bis-diethylenetriaminepentaacetic acid pamoicacid derivative obtained from 3-hydroxy-2-naphtalene methyl carboxylatethrough its reaction with hydrazine, exhibit a unique targetability tonecrotic tissues. Such pamoic acid derivatives however exhibit someshortcomings. First, solutions of these compounds with concentrationsuseful for medical applications are not colorless and, during long termstorage, may encounter significant discoloration of pharmaceuticalpreparations containing them. For instance, a 0.25 mmolar solution ofthe said gadolinium complex of the bis-diethylenetriaminepentaaceticacid pamoic acid derivative obtained from 3-hydroxy-2-naphtalene methylcarboxylate has a yellow-orange color and has an absorbance of 0.75 at400 nm. Secondly, despite their relatively high LD₅₀ values, significantside effects (e.g. myocarditis) were observed in animals having receivedan intravenous bolus injection of 1 mmole of the said gadolinium complexper kg body weight.

As a summary, the state of the art provides six different classes ofdisubstituted bis-indoles, being respectively the symmetrical1,3-disubstitution, 2,3-disubstitution, 2,5-disubstitution,3,5-disubstitution and 3,6-disubstitution and the disymmetrical2,2′,3,5′-substitution of a bis-indolyl basic structure. In addition tothe said six classes actually disclosed in the art, 1,2- and2,4-disubstituted indoles are also respectively available, which couldin principle be condensed, using coupling procedures standard in theart, in order to achieve the corresponding disubstituted bis-indoles.However, with very few exceptions, the bis-indoles bearing carboxylicacid ester substituents described so far are either symmetrical2,5disubstituted bis-indoles or the disymmetrical 2,2′,3,5′-substitutedbis-indoles.

In view of the prevalence of cerebral and myocardial infarction and ofother necrosis related phenomena, It would be of great importance toprovide contrast agents, in particular multipurpose contrast agents andtissue-specific contrast agents which would be in vivo effective for theidentification, localization and therapeutic follow-up of pathologicaltissue disorders, in particular those resulting from ischemic diseasesand space-occupying lesions, including necrosis, but which would notsuffer from the above-mentioned drawbacks (significant discolorationupon long-term storage, myocardial toxicity) of the pamoic acidderivatives known in the art. There is also a need in the art forpharmaceutical compositions including tissue-specific and/ormultipurpose contrast agents which are suitable for use in magneticresonance imaging and nuclear scintigraphy imaging technologies whilerequiring only low doses of the active agent. It is therefore the goalof the present invention to provide useful compounds meeting thesevarious criteria, as well as a suitable and cost-effective manufacturingroute for their synthesis.

SUMMARY OF THE INVENTION

Thus viewed from one aspect, the present invention provides new anduseful compounds having one of the formulae (I), (Ia) and (Ib) asspecified hereinafter, as well as methods for making them. The compoundsof general formula (I) are useful as vectors and agents in therapy andmedical diagnosis. Viewed from a further aspect, the invention providescompositions, use and methods for use comprising the compounds havingformula (I).

The present invention is mainly based on the unexpected finding thatuseful metal-complexable compounds may be obtained by first preparing2,3-disubstituted bis-indoles starting materials preferably bearing atleast one carboxylic acid reactive substituent (such as acid, acidhalide or acid ester) that is able either to directly condense with atleast a suitable chelating agent or to react with a spacing agent suchas a bis-amine or an amino-acid in order to achieve precursor compoundssuch as bis(indolecarboxylic acid hydrazides and analogues thereof whichthemselves are able to condense with at least a suitable chelatingagent. The substituted bis-indole derivatives thus produced, as well astheir enantiomers and their pharmaceutically acceptable salts, are ableto form complexes with radioactive and non-radioactive metals. Themetal-complexable substituted bis-indole derivatives and the metalcomplexes obtained therefrom constitute the active ingredients ofpharmaceutical compositions which are useful, among others, in thediagnosis or therapy of ischemic diseases or space-occupying lesions ina patient, for instance for imaging a Ussue in a mammal, or as anecrosis-avid agent or as a multipurpose contrast agent for variousorgans or parts of organs of a mammalian body, wherein the multipurposefunction includes at least one of a blood pool agent, a liver agent anda kidney agent. The present invention also relates to a method forgenerating an image of at least a part of the body of a mammal,comprising systemically or locally administering to the said mammal acontrast agent effective amount of such a metal-complexable substitutedbis-indole derivative or a metal complex or a pharmaceuticallyacceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows a synthesis procedure to obtain mono-reactiveanhydride bifunctional chelating agents which are useful in preparingthe metal-complexable bis-indole derivatives of the invention.

FIG. 2 shows the detailed formula of one exemplary metal-complexablebis-indole derivative according to the invention (the compound ofexample 8).

FIG. 3(A-D) illustrates the necrosis contrast agent function of oneexemplary metal-complexable bis-indole derivative according to theinvention by showing photographs of magnetic resonance images (MRI)obtained in a rat model of experimentally induced reperfused liverinfarction before (A) and after (B-D) intravenous injection of thecompound of examples 8. FIG. 3E shows a photograph of a macroscopichistological slice corresponding to the same MRI image, obtained aftersacrificing the same rat (arrows indicate necrotic liver lobe, S standsfor stomach).

FIG. 4(A-C) illustrates the necrosis contrast agent function of oneexemplary metal-complexable bis-indole derivative according to theinvention by showing magnetic resonance images of a pig with reperfusedmyocardial infrarction before (A) and after (B-C) intravenous injectionof the compound of example 8. FIG. 4D shows a photograph of a heartsection of the same pig.

FIG. 5 shows magnetic resonance angiographic images of a rabbit afterintravenous injection of a commercial contrast agent Gd-DTPA (A-C) as acomparative example, and (A′-C′) after intravenous injection of oneexemplary metal-complexable bis-indole derivative according to theinvention (the compound of example 8).

FIG. 6(A-E) shows magnetic resonance images of a rat with liverimplantation of rhabdomyocarcoma treated with radiofrequency ablationbefore (A) and after (B-E) intravenous injection of one exemplarymetal-complexable bis-indole derivative according to the invention (thecompound of example 8). FIG. 6F shows a photograph of a histologicalcross- section of the same rat.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with a first aspect of the present invention,metal-complexable substituted bis-indole derivatives are providedcomprising the structure shown in formula (I) hereunder:

wherein:

-   -   L represents a single bond or an optionally substituted linking        agent (Li) which covalently links together the carbon atoms        being respectively in positions 2 or 3 and 2′ or 3′ on the        heterocyclic rings of the indolyl groups;    -   R₁ and R₂ are optional substituents of any free position of the        phenyl rings of the indolyl groups;    -   R₃ is an optional substituent of the linking agent (Li);    -   q, p and r are integers indicating the number of the respective        substituents R₁, R₂ and R₃, provided that r is 0 when L is a        single bond;    -   C₁ and C₂ are optional metal-complexing substituents of the        heterocyclic rings of the indolyl groups;    -   m and n are integers indicating the number of the respective        metal-complexing substituents C₁ and C₂ and are each 0 or 1,        provided that the sum of m and n is at least 1.

Preferred embodiments of the metal-complexable substituted bis-indolederivatives of the present invention are those wherein at least one ofthe following limitations is met:

-   -   m is 1 and n is 1;    -   at least one of m and n is 1 and the metal-complexing        substituent C₁ or C₂ is in an α position with respect to the        single bond L or linking agent (Li);    -   m is 1, n is 1 and the metal-complexing substituents C₁ and C₂        are both in α positions with respect to the single bond L or        linking agent (Li);    -   the linking agent (Li) is selected from the group consisting of        a bridging at least divalent heteroatom, a disulfide bridge and        an optionally substituted alkylene group wherein the alkylene        chain may be interrupted by one or more heteroatoms; examples of        suitable at least divalent heteroatoms include oxygen and        sulfur; when sulfur is used as a heteroatom, (Li) may also be SO        or SO₂; examples of suitable alkylene groups include methylene,        ethylene and straight-chain or branched-chain alkylene groups        having from 3 up to 6 carbon atoms (such as trimethylene,        tetramethylene or hexamethylene), each hydrogen atom of the said        alkylene groups being possibly substituted with an R3        substituent such as defined hereinbelow in more detail; when the        alkylene chain is interrupted by one or more heteroatoms, the        latter are preferably selected from oxygen and sulfur, more        preferably oxygen, and the number of such heteroatoms is        preferably up to 6, a specific example being a polyethoxy chain.    -   preferably the linking agent (Li) is a methylene group        optionally substituted with one or two substituents R₃, the said        substituents R₃ being preferably non-functional, i.e non        reactive with chemical functions born by other parts of the        bis-indole derivatives of the invention, in particular by the        metal-complexing substituents thereof;    -   more preferably the linking agent (Li) is a methylene group        substituted with one or two substituents R₃, each substituent R₃        being an optionally substituted aryl or heteroaryl group or an        optionally substituted branched chain or straight chain alkyl        group having from 1 to 20 carbon atoms, wherein the substituents        on the said alkyl, aryl or heteroaryl group are preferably        substituents which are not easily oxidable, such as halogen        atoms (including fluorine, chlorine, bromine and iodine),        saturated or unsaturated hydrocarbon groups having 1 to 4 carbon        atoms (in particular alkyl groups such as methyl, ethyl,        n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl), alkoxy        groups having 1 to 4 carbon atoms (including methoxy, ethoxy,        propoxy and butoxy), cyano, carboxylic acid, sulfonic acid,        carboxylic acid ester wherein the ester group derives from an        alkyl group having 1 to 4 carbon atoms (such as above defined),        substituted or unsubstituted carboxylic acid amides CO—NR₄R′₄ or        substituted or unsubstituted amines NR₄R′₄ (wherein R₄ and R′₄        are each independently selected within radicals having the        meaning indicated for R₄ under the heading of chelating agents        hereinbelow); substituents on the aryl or heteroaryl group which        should preferably be avoided are hydroxyl, mercapto and groups        similarly susceptible of causing oxidation of the compounds of        the invention and consequently susceptible of causing a        significant discoloration of physiologically acceptable        solutions containing the said compounds; suitable examples of        the aryl group R₃ are phenyl, 4-biphenyl, 1-naphtyl, 2-naphtyl        and 2-fluorenyl; suitable examples of the heteroaryl group R₃        are pyridinyl, quinolinyl, isoquinolinyl, thiophenyl,        imidazolyl, pyrrolyl, furanyl, carbazolyl and N-(C₁–C₄)alkyl        substituted carbazolyl; the number of substituents on the aryl        or heteroaryl group R₃ may typically be from 0 to 3 or even        more, depending on the number of carbon atoms of the said group;    -   each of p, q and r is independently selected from integers        ranging from 0 to about 4.

Preferably, linking agents (Li) being a substituted methylene group maybe derived from aliphatic, aromatic or heteroaromatic aldehydes such as(in the following non-exhaustive list, use of the plural is meant toinclude all possible isomers) benzaldehyde, mono- and polyhalogenatedbenzaldehydes, cyano-benzaldehydes, substituted or non-substitutedaminobenzaldehydes, mono- and dinitro-benzaldehydes, mono- andpolyalkoxybenzaldehydes, mono- and polyalkylated benzaldehydes,carboxylated benzaldehydes, aryloxy-benzaldehydes,2-fluorenecarboxaldehyde, naphthaldehydes, alkoxy-naphthaldehydes,N-ethyl-3-carbazole-carboxaldehyde, 4-formylcinnamic acid,alkylthiobenzaldehydes, 2-formylbenzenesulfonic acid,methylformylbenzoate, acetaminobenzaldehyde, aryloxyalkylbenzaldehydes,acetamidobenzaldehyde, alkylsulfonylbenzaldehyde, propionaldehyde,butyraldehyde and so on.

Preferred C₁ and C₂ metal-complexing substituents are each independentlyrepresented by the formula -(Sp)_(s)-CA, wherein CA is a chelatingagent, (Sp) is a spacing agent being attached (i.e. covalently linked)both to the heterocyclic ring of the indolyl group and to the chelatingagent CA, and s is an integer selected from 0 and 1 (thus meaning thatthe spacing agent (Sp) is optional in the structure of themetal-complexable bis-indole derivative of the present invention). Inparticular, s may be 0 when an amino substituent is present on aheterocyclic ring of an indolyl group and the chelating agent CAcomprises a terminal functional group, such as carboxylic acid, acidhalide or acid ester that is able to readily react with the said aminosubstituent or when an amino substituent is present on the chelatingagent CA and a heterocyclic ring of an indolyl group comprises aterminal functional group, such as carboxylic acid, acid halide or acidester that is able to readily react with the said amino substituent.However, when introducing an amino substituent on the heterocyclic ringof an indolyl group proves to be difficult, then preferably s is equalto 1.

In the previous definition of C₁ and C₂, the structure of the chelatingagent CA may comprise:

-   -   one or more thiol bearing moieties such as for example        bisamine-bisthiol, bisamine-bisoxime, monomercapto-triamide,        diamide-dithiol, monoamine-monoamide-dithiol, tetramine,        monoamine-diamide-monothiol, monoamine-monothioether-dithiol,        monoamine-monothiol, monoamide-diamine-monothiol and diphosphine        based moieties (for reasons of stability of the complex formed        with certain metals, this type of chelating agent is preferred        namely when the metal is technetum-99m, rhenium-186 or        rhenium-188); and/or    -   one or more structural elements with the following formulae

-   -    wherein Z′ is a radical selected from the group consisting of        phosphonomethyl (—CH₂PO₃HR₄), carboxymethyl (—CH₂COOR₄) and its        derivatives (—CHR₅COOR₄), carboxyethyl (—CH₂CH₂COOR₄) and its        derivatives (—CHR₅CH₂COOR₄ and —CH₂CHR₅COOR₄); Z″ is hydrogen or        a radical in the meaning of Z′ or hydroxyethyl —CH₂CH₂OH or its        derivatives —CHR₅CH₂OH and —CH₂CHR₅OH), i.e. for instance the        nitrogen atom together with Z′ and the optional Z″ stand for        iminoacetic acid, iminopropionic acid, iminodiacetic acid,        iminodipropionic acid, iminoacetic propionic acid, hydroxyethyl        iminoacetic acid and hydroxyethyl iminopropionic acid; R₄ is        selected from hydrogen and optionally substituted C₁–C₂₀        branched chain or straight chain alkyl groups or C₆–C₂₀ aryl        groups or C₆–C₂₀ alkylaryl groups, wherein the substituents on        the alkyl, aryl or alkylaryl group may be for instance halogen        atoms (including fluorine, chlorine, iodine and bromine), nitro,        carboxy, amino, aminyl, amido or sulfono and wherein the number        of such substituents may be from 0 to 3 or even more, depending        on the number of carbon atoms of the said group; and R₅ is        selected from groups in the meaning of R₄ or bonding groups        comprising —CO— and/or —NH—, carboxy (—COOR₄), or radicals        having the formula        —(CH₂)₀₋₅—(C₆H₄)₀₋₁—(O)₀₋₁—(CH₂)₀₋₁—(C₆H₄)₀₋₁—(O)₀₋₁-M (II), or        the formula        —(CH₂)₀₋₅—(C₆H₁₀)₀₋₁—(O)₀₋₁—(CH₂)₀₋₅—(C₆H₄)₀₋₁-M (III) wherein        the integers appearing as subscripts of the hydrocarbylene or        oxygen bonding groups indicate the minimum and maximum numbers        of such groups and M is a group in the meaning of R₄, a        —CH₂CH₂COOR₄ group, a bonding group comprising —CO— and/or —NH—,        or a saturated, unsaturated or aromatic heterocyclic group        comprising one or more heteroatoms (such as e.g. furfuryl and        imidazolyl) and optionally substituted by up to 3 independent        substituents being for instance such as listed hereinabove.

In accordance with the previous definitions, a first class of preferredchelating agents CA may be represented by one of the following formulae:

wherein

-   -   a is an integer ranging from 0 to 6,    -   each V independently represents an optionally substituted        saturated, unsaturated or aromatic organylene group, such as        phenylene, diphenylene, straight-chain or branched-chain        hydrocarbylene group wherein the chain or part of it may form a        cyclic or heterocyclic ring and wherein the hydrocarbylene group        may also contain a phenylene, oxygen, sulfur, aminyl,        N-substituted aminyl, carbonyl or thiocarbonyl group; the        optional substituents of the said organylene group being        preferably such as listed hereinabove with reference to the        substituents of an aryl or heteroaryl group R₃ of the linking        agent (Li) or being radicals having the formulae (II) or (III)        hereinabove or being hydroxyl or mercapto groups, and    -   each Z independently represents hydrogen or —CHR₅-Q or        —CO—CHR₅-Q or —CHR₅—CHR′₅-Q, wherein Q is a bonding group        selected from —CO—, —NH—, carboxy (—COOR₄), phosphono (—PO₃HR₄),        amido (—CONR₄R′₄), hydroxy, alkoxy and aryloxy —OR₄, thiol,        mercapto—SR₄, hydrazo (—CO—NH—NHR₄) and saturated, unsaturated        or aromatic heterocyclic groups comprising one or more        heteroatoms (such as e.g. furfuryl, pyridyl and imidazolyl) and        optionally substituted by up to 3 independent substituents being        preferably non-easily oxidable substituents such as listed        hereinabove with reference to the substituents of a heteroaryl        group R₃, and further wherein R₄ and R₅ are as defined-        hereinabove and R′₄ and R′₅ have respectively the same meaning        as R₄ and R₅.

Among the chelating agents CA of formula (IV) which comprise one or morethiol or mercapto groups, V is preferably —CH₂—CH₂—, —CO—CH₂—,—(CH₂)₂—NH—(CH₂)₂— or —(CH₂)₂—N(-Z)-(CH₂)₂—. In this case, preferred arechelating agents CA having one or more aminyl Z substituentsindependently selected from thioethyl and derivatives thereof(—CH₂CH₂SR₄), —COCH₂SR₄, thiomethyl and derivatives thereof (—CH₂SR₄),the remaining aminyl Z substituents being preferably selected fromhydrogen, phosphonomethyl, carboxymethyl and derivatives thereof,carboxyethyl and derivatives thereof, hydroxyethyl and derivativesthereof such as previously defined (and including —CH₂CH₂OR₄).Additionally, one or more of the remaining aminyl Z substituents mayrepresent a heterocyclic group such as previously indicated. Suitableexamples thereof include the following:

-   -   derivatives of a mercaptoacetyl tripeptide such as        mercaptoacetyl triglycine,    -   an ethylene diamine dioxime tetraligand, a butylene diamine        dioxime tetraligand or a propylene diamine dioxime tetraligand        bearing one or more alkyl substituents (the said alkyl        substituent being a group including from 1 to 8 carbon atoms)        such as hexamethylpropylene diamine dioxime (HMPAO),    -   ethylene dicysteine, ethylene cysteine cysteamine,        cysteinylglycine cysteine, bismercaptoacetyldiaminopropionic        acid, bismercaptoacetyidiaminosuccinic acid,        bismercaptoacetyidiaminobutyric acid,        N-(mercaptoacetylaminoethyl)cysteine, dimercaptosuccinic acid,        dimercaptopropionic acid, cysteine, cysteamine,        diphosphinopropionic acid, and derivatives thereof wherein one        or more thiol functions are protected by a suitable R₄ group        such as defined hereinabove.        The latter chelating agents are preferred namely when the        complexing metal used is technetium-99m, rhenium-186 or        rhenium-188.

Among the chelating agents CA of formulae (IV) and (V) hereinabove whichdo not comprise a thiol function, V is preferably —CH₂—CH₂—, —(C₅H₈)—,—(C₆,H₁₀)—, —(CH₂)₂—NH—(CH₂)₂— or —(CH₂)₂—N(-Z)-(CH₂)₂—, and the integera in formula (V) is preferably 0 or 1. In this case, preferred chelatingagents have two or more, preferably three to five, aminyl Z substituentseach independently selected from the group consisting of carboxymethyl(—CH₂COOR₄) and derivatives thereof (—CHR₅COOR₄), carboxyethyl(—CH₂CH₂COOR₄) and derivatives thereof (—CHR₅CHR′₅COOR₄). Remainingaminyl Z substituents are preferably each independently selected fromhydrogen, phosphonomethyl, carboxymethyl and derivatives thereof,carboxyethyl and derivatives thereof, hydroxyethyl and derivativesthereof (such as previously defined), methylamido (—CH₂CO—NH₂) orderivatives thereof (—CH₂CO—NH—R₄ and —CH₂CO—NR₄R′₄), ethylamido(—CH₂CH₂CO—NH₂) or derivatives thereof (—CH₂CH₂CO—NHR₄ and—CH₂CH₂CO—NR₄R′₄), methylhydrazido (—CH₂CO—NH—NHR₄ and—CH₂CO—NH—NR₄R′₄), ethylhydrazido (—CH₂CH₂CO—NH—NHR₄ and—CH₂CH₂CO—NH—NR₄R′₄). Additionally, one or more of the remaining aminylZ substituents can represent a straight-chain or branched-chainsaturated or unsaturated alkyl group optionally substituted by halogenatoms and/or functional groups and optionally comprising a saturated,unsaturated or aromatic heterocyclic group comprising one or moreheteroatoms and optionally bearing up to 3 independently selectedsubstituents, such as for instance (furfuryl)alkyl,(hydroxyfurfuryl)alkyl, (imidazolyl)alkyl, (methylimidazolyl)alkyl,benzyl, benzyloxymethyl, 4-carboxymethoxybenzyl, 4-methoxybenzyl,4-ethoxy-benzyl, 4-butoxybenzyl, 4-benzyloxybenzyl,4-(4-methyloxybenzyloxy)-benzyl, methyl, propyl, isopropyl, n-butyl,sec-butyl, tert-butyl, pentyl, cyclopentyl, cyclohexyl, hydroxymethyl,2-hydroxyethyl, 2-hydroxy-1-(hydroxymethyl)ethyl, 3-hydroxypropyl,2-hydroxyisobutyl, 2,3-dihydroxypropyl, hydroxybutyl,4-hydroxy-2-methylbutyl, 2,3,4-trihydroxybutyl and2,3,4,5,6pentahydroxyhexyl.

Chelating agents CA of formulae (IV) and (V) which do not comprise athiol function preferably have carboxymethyl groups (—CH₂COOR₄ whereinR₄ is as defined above) as the predominant aminyl Z substituents.Suitable examples thereof include:

-   -   ethylenediaminetetraacetic acid (usually referred as EDTA),        diethylerie triaminopentaacetic acid (DTPA),        trans-1,2-cyclohexanediamine tetraacetic acid (CDTA),        1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA),        1,4,7-triazacyclononanetriacetic acid,        1,4,8,11-tetraazacyclotetradecanetetra-acetic acid (TETA),        ethyleneglycol-O,O′-bis(2-aminoethyl)tetraacetic acid (EGTA),        N,N-bis(hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED),        triethylenetetramine hexaacetic acid (TTHA), hydroxyethyidiamine        triacetic acid (HEDTA) and 1,5,9-triazacyclo-dodecanetriacetic        acid, and    -   analogues of the above compounds wherein one or more        carboxymethyl groups are replaced by hydrogen and/or by another        aminyl Z substituent in such a way that Z together with the        aminyl group to which it is attached comprises one of the        following: aminoethanol, 2-amino-2-ethyl-1,3-propanediol,        3-methyl-1-butamine 6-amino-2-methyl-2-heptanol,        2-(2-aminoethoxy)ethanol, 1-(3-aminopropyl) imidazole,        4-(3-aminopropyl)morpholine, 1-(3-aminopropyl) -2-pipecoline,        1-(3-aminopropyl)-2-pyrrolidinone, 1-(2-aminoethyl)pyrrolidine,        1-(2-aminoethyl)piperidine, 2-(2-aminoethyl)pyridine,        2-(aminomethyl)pyridine, 1-(2-aminoethyl)pyrrolidine,        4-(2-aminoethyl)morpholine,        4-(2-aminoethyl)-1-methylpyrrolidine, (aminomethyl)cyclopropane,        2-(aminomethyl)pyridine, 3-(aminomethyl)pyridine,        2-(aminomethyl)-1-ethylpyrrolidine, furfurylamine,        tetrahydrofurfurylamine, 2-(aminomethyl)-tetrahydropyran,        2-(aminomethyl)-1,3-dioxolane,        2-(aminomethyl)-1-methylimidazole, N-(2-aminoethyl) piperidine,        N-(2-aminoethyl)morpholine, 3-(2-aminoethyl)indole, naturally        occurring amino-acids such as glycine (equivalent to a        carboxymethyl Z substituent), leucine, isoleucine, isoleucinol,        alanine, β-alanine, valine, tyrosine, serine or threonine, and        analogues thereof such as their carboxamide, hydrazide or ester.        Suitable examples of such analogues include N-(2-hydroxyethyl)        diethylenetriamine N,N′,N″,N′″ tetraacetic acid and        N-(2-hydroxyethyl) ethylenediamine N,N′,N″ triacetic acid.

Among the chelating agents CA of formulae (IV) and (V), especiallypreferred are those having structures represented by any of thefollowing formulae 10a–10g and 11a–11b:

wherein the arrow indicates the bonding position of the said chelatingagent to the ring system A and/or B or to the optional spacing agent(Sp), and wherein R_(Z) has the same meaning as described for theradical R₄ hereinabove.

Other classes of alternatively suitable chelating agents CA which may beused in the present invention have been extensively described in theprior art, for instance in U.S. Pat. Nos. 6,221,334, 6,056,939,5,556,939, 5,527,885, 5,326,856, 5,220,000, 5,202,451, 5,196,515,5,164,176, 5,585,468, 4,861,869, 5,436,352, 6,184,361, 5,808,003,5,756,825 and 5,632,969, the content of which is incorporated herein byreference.

The optional spacing agent (Sp) which may be present in themetal-complexing substituents C₁ and/or C₂ is preferably a radicalrepresented by the formula —X)_(t)-(Sp′)-, wherein t is 0 or 1, (Sp′) isderived from a molecule bearing:

-   -   at one end a first functional group (i), such as an amine,        suitable for reacting with a carboxylic acid function (e.g.        acid, acid halide or acid ester), for instance with the        carboxylic acid function preferably present on the heterocyclic        rings of the indolyl groups of a suitable 2,3-disubstituted        bis-indole precursor compound bearing at least one carboxylic        acid (halide or ester) or amino substituent; and    -   at the other end a second functional group (ii) suitable for        reacting with the chelating agent CA of interest, and        X is a carboxy group or a primary or secondary amino group.

Based on the above definition, the person skilled in the art is readilyable to determine one or more suitable spacing agents (Sp) once thechelating agent CA has been selected. In practice, preferred spacingagents (Sp) are radicals derived from bis-amines, such as preferablyhydrazine, or from amino-acids, such as detailed hereinafter in thecontext of the methods for producing the novel compounds of theinvention.

The optional substituents R₁ and/or R₂ which may be present on thephenyl ring of the indolyl groups may be each independently selected forinstance from the following list:

-   -   halogen atoms such as fluoro, chloro, iodo and bromo,    -   straight chain alkyl groups of 1 to 6 carbon atoms such as        methyl, ethyl, n-propyl, n-butyl, n-pentyl and n-hexyl,    -   branched chain alkyl groups of 3 to 8 carbon atoms such as        isopropyl, isobutyl, tert-butyl, isoamyl, methylpentyl,        2-ethylhexyl and the like,    -   cycloalkyl groups of 3 to 7 carbon atoms, in particular        cyclopentyl and cyclohexyl,    -   straight chain and branched chain alkoxy groups of 1 to 6 carbon        atoms such as methoxy, ethoxy, propyloxy, buyloxy and amyloxy,    -   trifluoromethyl,    -   cyano,    -   carboxylic acid,    -   sulfonic acid,    -   carboxylic acid ester (wherein the ester group derives from an        alkyl group having 1 to 4 carbon atoms (such as above defined),        and    -   substituted or unsubstituted amines NR₄R′₄ (wherein R₄ and R′₄        have the meaning indicated hereinbefore).

Of particular importance here is the fact that, alike for the optionalsubstituents of an aryl or heteroaryl group R₃, the R₁ and/or R₂substituents are preferably substituents which are not easily oxidable.Therefore, hydroxyl and mercapto groups and other functional groupssimilarly susceptible of causing oxidation of the compounds of theinvention and consequently susceptible of causing a significantdiscoloration of physiologically acceptable solutions containing thesaid compounds should preferably be avoided.

As previously set forth, the single bond or linking agent L covalentlylinks together the carbon atoms being respectively in positions 2 or 3and 2′ or 3′ (using conventional nomenclature rules) on the heterocyclicrings of the indolyl groups of the bis-indole derivatives of theinvention. This means that within the broad chemical structure shown informula (I), three distinct sub-families of derivatives may berecognized as follows:

-   -   a sub-family wherein L covalently links the carbon atoms being        respectively in positions 3 and 3′, having the structure shown        in formula (Ia) hereunder:

wherein L, C₁, C₂, R₁, R₂, R₃, p, q and r are as defined hereinabove,

-   -   a sub-family wherein L covalently links the carbon atoms being        respectively in positions 2 and 2′, having the structure shown        in formula (Ib) hereunder:

wherein L, C₁, C₂, R₁, R₂, R₃, p, q and r are as defined hereinabove,and A and B designate the heterocyclic rings of the indolyl groups, and

-   -   a sub-family wherein L covalently links the carbon atoms being        respectively in positions 2 and 3′.

Exemplary useful and readily available metal-complexable substitutedbis-indole derivatives corresponding to formula (Ia) are for instanceselected from the group consisting of:

-   -   [{2-[N′-(3-{[2-(N′-{2-[(2-{[2-bis(carboxymethylamino)-ethyl]carboxymethyl-amino}ethyl)carboxymethylamino]-acetyl}hydrazinocarbonyl)-1H-indol-3-yl]-phenyl-methyl}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-acetic        acid,    -   [{2-[N′-(3-{[2-(N′-{2-[(2-{[2-bis(carboxymethyl-amino)-ethyl]carboxymethyl-amino}-ethyl)carboxymethyl-amino]-acetyl}-hydrazinocarbonyl)-1H-indol-3-yl]-methyl}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-acetic        acid,    -   {4,7-bis-carboxymethyl-10-[({3-[(2-{N′-[(4,7,10-tris-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetyl]-hydrazinocarbonyl}-1H-indol-3-yl)-methyl]-1H-indole-2-carbonyl}-hydrazino)-2-oxo-ethyl]-1,4,7,10-tetraaza-cyclododec-1-yl}-acetic        acid,    -   {4,8-bis-carboxymethyl-11-[({3-[(2-{N′-[(4,8,11-tris-carboxymethyl-1,4,8,11-tetraaza-cyclotetradec-1-yl)-acetyl]-hydrazinocarbonyl}-1H-indol-3-yl)-methyl]-1H-indole-2-carbonyl}-hydrazino)-2-oxo-ethyl]-1,4,8,11-tetraaza-cyclotetradec-1-yl)acetic        acid,    -   enantiomers and pharmaceutically acceptable salts (including        alkaline salts and ammonium salts) thereof.

Such salts include sodium and potassium salts and tertiary ammoniumsalts NR₄R′₄R″₄R′″₄ (wherein R₄, R′₄, R″₄ and R′″₄ are eachindependently selected within the meaning of R₄ provided hereinaboveunder the heading of radicals Z′ of the chelating agents). Moreparticularly useful are the sodium salts of:

-   -   [{2-[N′-(3-{[2-(N′-{2-[(2-{[2-bis(carboxymethyl-amino)-ethyl]carboxymethylamino}-ethyl)carboxymethyl-amino]-acetyl}-hydrazinocarbonyl)-1H-indol-3-yl]-phenyl-methyl}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-acetic        acid, and    -   [{2-[N′-(3-{[2-(N′-{2-[(2-{[2-bis(carboxymethyl-amino)-ethyl]carboxymethylamino}-ethyl)carboxymethyl-amino]-acetyl}-hydrazinocarbonyl)-1H-indol-3-yl]-methyl}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-acetic        acid.

The term “enantiomers”, as used herein, means each individual opticallyactive form of the compound of the invention, having an optical purity(as determined by methods standard in the art) of at least 80%,preferably at least 90% and more preferably at least 98%.

In the following part of the description, it should be understood that,although alternative chemical routes may exist, a suitable and preferredmethod for preparing the metal-complexable substituted bis-indolederivatives corresponding to formula (I) involves the following steps:

-   -   (a) a bis-indole compound, wherein the carbon atoms being        respectively in positions 3 and 3′ or 3 and 2′ or 2 and 2′ on        the heterocyclic rings of the indolyl groups are covalently        linked through L, i.e. through a single bond or an optionally        substituted linking agent (Li), the said bis-indole compound        optionally comprising one or two reactive groups in α-position        with respect to L, is first prepared by a coupling reaction        involving (i) one mono-indole or two different mono-indoles each        optionally comprising one reactive group, the said reactive        group being preferably a carboxylic group (for instance a        carboxylic acid, a carboxylic acid halide or a carboxylic alkyl        ester group) or an amine, and (ii) an aldehyde comprising the        moiety (Li),    -   (b) at least one reactive group, if not already present on the        bis-indole compound from step (a), is introduced onto the        heterocydic ring of at least one indolyl group of the said        bis-indole compound, in α-position with respect to L, the said        reactive group being preferably a carboxylic group (for instance        a carboxylic acid, a carboxylic acid halide or a carboxylic        alkyl ester grou) or an amine,    -   (c) the reactive group(s) mentioned in step (a) or step (b) is        (are) optionally reacted, usually by a coupling reaction, with        at least one spacing agent Sp such as previously defined (i.e.        bearing at least two functional end groups) and    -   (d) the second functional end group of the spacing agent Sp is        reacted, usually by a coupling reaction, with a suitable        chelating agent CA, such as previously defined, or a chelating        agent precursor being able to introduce the desired chelating        moiety into the metal-complexable derivative of the invention.

Whilst the mono-indoles and the coupling reaction conditions of step (a)are well known in the art, the same cannot be said of the2,3-disubstituted bis-indole intermediates or precursor compoundsobtained after step (b) and/or after step (c), which are novel chemicalcompounds having utility in the preparation of the metal-complexablederivatives of the invention.

Therefore, in accordance with a second aspect of the invention, a firstclass of novel precursor compounds for the metal-complexable substitutedbis-indole derivatives of formula (I) consists of compounds obtainedafter step (c), being 2,3-disubstituted bis-indole compounds optionallyhaving one or more spacing agents (Sp) attached, in α-position whichwith respect to L, to the heterocyclic ring(s) of the indolyl group(s).

Specific examples of this first class of novel precursor compounds arethe bis-hydrazides(3′-{[(2-hydrazinocarbonyl)-1H-indol-3-yl]-phenyl-methyl-1H-indole-2-carbonyl)-hydrazineand(3′-{[(2-hydrazinocarbonyl)-1H-indol-3-yl]-methyl}-1H-indole-2-carbonyly)-hydrazine,their indol-2-yl isomers (i.e., using trivial names,3,3′-benzylidenebis(indole-2-carboxylic acid hydrazide) and3,3′-methylenebis (indole-2-carboxylic acid hydrazide) and analoguesthereof. The term “analogues” makes reference to similar compoundswherein (i) the phenyl ring of each indolyl group may be independentlysubstituted with substituents R₁ and/or R₂ such as previously definedfor the derivatives of formula (I) and/or wherein (ii) the methylene orbenzylidene bridging group between the indolyl groups may be replacedwith a single bond or with any other linking agent (Li) such aspreviously defined for the derivatives of formula (I) and/or wherein(iii) hydrazine is replaced by another bis-amino or amino-acid radical,as defined hereinafter.

Altogether, this first class of novel precursor compounds comprise thestructure shown in formula (VIII) hereunder:

wherein:

-   -   L, R₁, R₂, R₃, p, q and r are as defined hereinabove, and    -   X and each of Sp₁ and Sp₂ respectively have the meaning of X and        Sp′ given hereinabove.

This first class of precursor compounds may be readily prepared duringstep (c) by reacting a spacing agent—such as hydrazine, a bis-amine oran amino-acid—with a bis-indole compound comprising at least one groupreactive with the first functional end group of the said spacing agentSp, for instance a 2,3-disubstituted carboxylated bis-indole such as3′-{[(2-alkyloxycarbonyl)-1H-indol-3-yl]-phenyl-methyl}-1H-indole-2-carboxylicacid ethyl ester, their indol-2-yl isomers and analogues thereof wherein(i) the phenyl ring of each indolyl group may be independentlysubstituted with substituents R₁ and/or R₂ such as previously definedand/or wherein (ii) the methylene or benzylidene bridging group betweenthe indolyl groups may be replaced with a single bond or with any otherlinking agent (Li) such as previously defined. The latter may bereferred as a second class of novel precursor compounds, beingintermediates useful for the preparation of the precursors of the firstclass. These 2,3-disubstituted carboxylated bis-indoles in turn can besuitably prepared either according to step (b) or by another procedurecomprising coupling one or more mono-indole compounds, at least one ofthem bearing a carboxylic group-containing substituent (for instance acarboxylic acid, a carboxylic acid halide or a carboxylic alkyl estergroup) or an amino-containing substituent (for instance an alkyleneamine —(CH₂)_(n)—NH₂,) in α-position or β-position (depending whether acompound of formula (Ia) or a compound of formula (Ib) is desired in thefinal step) with respect to the nitrogen atom of the indolyl group, bymeans of a known coupling agent. Suitable coupling agents for thispurpose include aromatic and heteroaromatic aldehydes (an extensive listof which has been provided hereinbefore) or formaldehyde, thus yieldinghomodimeric or heterodimeric (i.e. symmetric or not) bis-indolecarboxylic acid or bis-indole amino compounds, depending on whether oneor two different mono-indole compounds were used in the said couplingreaction.

The second class of precursor compounds comprise the structure shown informula (IX) hereunder:

wherein L, R₁, R₂, R₃, p, q, X and r are as defined hereinabove.

A spacing agent (Sp) can be introduced into the bis-indole moleculeduring step (c) in various ways. These include for example themodification of one or more available W substituents as shown in formula(VII) hereinafter or the introduction of one or more new W substituentsand/or combinations thereof. Again, use can be made of compoundsreferred to as homobifunctional spacing agent precursors (optionallywith one or more function(s) in a protected form) or theirheterobifunctional analogues in an optionally protected form. Forinstance, an α-C(═O)—NH—NH₂ substituent can be derived by coupling anα-carboxyl group with tert-butyl carbazate and a catalytic amount of acondensing agent such as dicyclohexylcarbodiimide,N-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline,N,N′-carbonyldiimidazole,2-isobutoxy-1-isobutoxy-carbonyl-1,2dihydroquinoline or the like,followed by deprotection in order to make the hydrazide available forfurther reaction. The same substituent can also be obtained byhydrazinolysis of a α-carboxyl alkyl ester group with hydrazine.Hydrazinolysis, aminolysis and the like are appropiate alternative wellknown procedures for reactions involving condensing agents andprecursors bearing protected functional groups which necessitatedeprotection.

Analogues of the bis-hydrazide precursors of the first class arecompounds for the preparation of which hydrazine is replaced with:

-   -   a bis-amine for instance selected from alkyl diamines having the        formula NH₂—(CH₂)₂₋₁₂—NH₂ including 1,2-diaminoethane,        1,5-diaminopentane 1,6-diaminohexane, 1,7-diaminoheptane        1,2-diaminododecane and the like, alkyl diamines comprising one        or more hetero atoms such as for example        1,8-diamino-3,6-dioxaoctane, 1,5-diamino-3-oxapentane; alkyl        diamines comprising one or more optionally protected functional        groups such as for example lysine and lysine; cyclic bis-amines        such as for example piperazine and derivatives thereof; or    -   an amino-acid, whether naturally-occurring or not, including a        straight chain or branched chain hydrocarbon group with 1 to 6        carbon atoms.

Once a precursor compound of the first class is available, then achelating agent CA can be introduced during step (d) of the preparationprocess, either directly or indirectly by making use of a precursor CAcompound. As used herein, precursor CA compounds include but are notrestricted to the well-known bi-functional chelating agents which,beside the functional groups (most often shielded by protective groups)necessary for metal complexation, bear a functional group (most often inan activated form, e.g. carboxylic acid esters comprising an appropriateleaving group) that can be specifically used in conjugation reactions.Accordingly, a bi-functional chelating agent is useful for introducingone or more CA functions into the bis-indole molecule. This can beaccomplished by conjugating the bi-functional chelating agent directlyto an atom which is part of the indolyl groups or the spacing agent(Sp), for example the nitrogen atom of a primary or secondary amine.

When step (d) involves an amine on the precursor compound of the firstclass (i.e. a bis-indole/spacing agent conjugate) or in case thebis-indole contains one or two amino-containing substituents on theheterocyclic rings of the bis-indoles, then a carboxylic acid group (orits activated form) of the chelating agent CA is usually involved in thecoupling procedure, as is the case for instance in bi-functionalchelating agents bearing an intra-molecular anhydride such as thosederived from an iminodiacetic acid molecule portion (—N(CH₂COOH)₂),Exemplary compounds of this kind comprise the bis-anhydrides of EDTA,DTPA and the like (such as disclosed in French Patent No. 1.548.888) aswell as the DTPA monoanhydride mono-ethyl ester. The latter compound maysuitably be prepared from DTPA monoethyl ester by reacting with aceticanhydride in the presence of pyridine. Other bi-functional DTPAderivatives are known from the literature (see e.g. J. Org. Chem. (1990)55:2868 and U.S. Pat. No. 5,514,810).

When step (d) involves an amine on the chelating agent CA, then one ortwo carboxylic acid groups (or their activated form) of the precursorcompound of the first class (i.e. a bis-indole/spacing agent conjugate)may be involved in the coupling procedure.

For the preparation of DTPA monoethyl ester and related compounds,reference is now made to FIG. 1. The present invention includes a novelmethod for obtaining monoreactive mono-anhydrides comprising thestructural formula VI in FIG. 1 via an intermediate which facilitatestheir purification. Such intermediate may be a monobenzyl ester obtainedby reacting the corresponding bis-anhydride having the formula 1 shownat top of FIG. 1 with benzyl alcohol in the presence of a suitablereagent such as for example water, ammonia, primary or secondary amines,hydrazides or alcohols, such as detailed hereinafter.

In the formulae 1 and VI of FIG. 1:

-   -   the indicia s, t, u and v are each independently selected        integers from 1 to 3, provided that the sum of s and t is not        above 4 and that the sum of u and v is not above 4;    -   V is an organylene group such as described in detail in respect        of formulae (IV) and (V) hereinabove; and    -   substituents U_(x) and U_(y) are freely but mutually exclusively        selected from either the group comprising —OH and —O⁻Cat⁺,        wherein Cat⁺ is an organic cation, e.g. a carboxylic acid        ammonium, sodium, calcium or potassium salt, or the group        comprising aminyl radicals of formula —NH₂ or substituted        derivatives thereof and ether-type radicals, i.e. U_(x) and        U_(y) together with the neighbouring carbonyl group may be a        carboxylic acid, a carboxylic acid salt, an (optionally N,N- or        N-substituted) amide, a hydrazide or a bis acylhydrazide group.

The bis-anhydride analogue of formula 1 in FIG. 1, obtained for instanceaccording to the method of French Patent No. 1,548,888, is dissolved(with optional heating) in a suitable anhydrous solvent (for exampledimethylformamide) in the presence of a base which does not react withanhydrides (for example a tertiary amine such as triethylamine). Next,equimolar amounts of an alcohol preferably comprising one or morearomatic groups (for example benzyl alcohol) and a suitable reagent areadded. When a maximal number of carboxylic groups is desired, then thesaid reagent is preferably a lower alcohol (such as for example methylor ethyl alcohol) which can easily be removed by hydrolysis. When, onthe other hand, a transformed carboxylic moiety is desired, then thereagent is preferably selected from amino, hydrazo and hydrazidocompounds. Even when reaction conditions are optimal with respect totemperature, dilution and method of introduction of reactants, asubstantial amount of by-products will be present. Hence, a second stepinvolves the physical separation of the major reaction products.Selecting an alcohol comprising one or more aromatic groups in the firststep enables purification to proceed under milder conditions, i.e. forexample non-hydrolytic conditions with respect to lower alkyl esters) byusing methods that allow separation based on differences inhydrophobicity and type of hydrophobic interaction. These methodstypically include extraction-based procedures, chromatographicprocedures using reversed phase chromatography on alkylated solidsupports, adsorption chromatography on polymeric supports, and so on.Fractions containing the essentially pure compound of formula 2 in FIG.1, wherein the B substituents mutually exclusively represent either aproton or the non hydroxyl portion of the alcohol used as the reagent ofthe previous step, are further processed by selective removal of the Bsubstituents in step 3 in order to afford a compound (having formula 2in FIG. 1) comprising a shielded terminal imino bis alkylcarboxylic acidgroup and an unprotected counterpart which, in final step 4 is convertedinto the desired monoreactive monoanhydride of formula VI.

For some applications (e.g. preparation of compounds comprising alkalineand/or acidic labile groups) it may be beneficial to accomplish couplingwith DTPA comprising unprotected carboxyl group(s) (e.g. devoided ofester groups) because removing protective groups often necessitatesstringent conditions (such as alkaline hydrolysis of the esterfunctions), as shown in one of the following examples.

To summarize, a chelating agent function CA can be introduced into aprecursor compound of a bis-indole derivative of formula (I) at anystage of the synthesis. In particular, it may be introduced previouslyto condensing the indolyl groups, although it is more often advantageousand easier to introduce it at a later stage, as explained hereinabove.Exemplary novel precursor compounds according to the invention comprisethe structure shown in formula (VII) hereunder:

wherein:

-   -   L, m, n, p, q and r are all as defined in formula (I)        hereinabove, and    -   W₁ and W₂ are optional substituents which, optionally after        adequate chemical modification, enable the attachment of        metal-complexing substituents such as C₁ and C₂ of formula (I)        respectively, and being at position 2 or 2′ (when L is at        position 3 or 3′) or at position 3 or 3′ (when L is at position        2 or 2′) of each indolyl group, i.e. in α position with respect        to L.

The precursor compounds of formula (VII) are preferably symmetric withregard to the indolyl groups, however due to the occurrence of possiblydifferent substituents on the phenyl ring of the indolyl groups, andpossibly due to the linking agent (Li), they may also be asymmetric.

After reaction with suitable metal-complexing substituents (i.e.chelating agents optionally separated from the indolyl group by aspacing agent), including optional chemical modification of the W₁and/or W₂ substituents, substituted bis-indole derivatives having thestructure shown in formula (I) are obtained from the first class ofnovel precursor compounds of this invention. For this purpose, a (secondclass) precursor compound bearing a carboxylic ester function (such ase.g. a3′-{[(2-alkyloxycarbonyl)-1H-indol-3-yl]-phenyl-methyl}-1H-indole-2-carboxylicacid ester) can be converted via aminolysis (e.g. by means ofethylenediamine, piperazine, 2-methylpiperazine or other suitablediamines, including those cited above under the heading homobifunctionalagents) or hydrazinolyis into an intermediate which readily reacts witha chelating agent precursor, e.g. with an anhydride (including DTPAmonoethyl ester monoanhydride) or with any kind of in situ activated ormono-reactive bifunctional chelating agent. Accordingly chelating groupsCA, including those cited in formulae 10a to 10g, 11a and 11bhereinabove, can be easily introduced into the final bis-indolederivative having the structure of formula (I).

According to the above-mentioned procedures, a multitude of specificsubstituted bis-indole derivatives, each embodying the structure offormula (I), may be produced which may exhibit some quantitativedifferences with respect to their properties in medical applications,such as blood clearance (ranging from relatively fast to relativelyslow), elimination from the body (predominantly by kidney or shifted tohepatobiliary secretion), plasma protein binding (from low to high),etc. Labeling/complexation of the substituted bis-indole derivativesaccording to formula (I) can be accomplished, using methods well knownin the art, by chelation with radioactive or non-radioactive metal ions,preferably with ions of an element with an atomic number selected from21 to 32, 37 to 39, 42 to 44, 49, 50 or 57 to 83 such as for example:

-   -   Mn, Fe or Gd (with respect to non-radioactive metals), and    -   ^(99m)Tc, ¹¹¹In, ⁶⁷Ga, ⁹⁰Y, ¹⁸⁸Re, ¹⁸⁶Re and ¹⁶³Dy (with respect        to radioactive metals).

Chelation with metal ions can be performed by methods well documented inthe literature, i.e. at any stage of the production, although most oftenin the final step. When protected functional groups are present in themetal-complexing substituents, they may be partly or completelydeprotected prior to metal chelation. Ionizable groups not involved inmetal complexation may be optionally neutralized by acidic or basiccounter-ions or by (inorganic and/or organic) compounds bearingionizable acidic and/or basic groups. Remaining acidic protons, i.e.those that have not been substituted by the metal ion, can optionally becompletely or partially replaced by cations of inorganic or organicbases, basic amino-acids or amino-acid amides. Suitable inorganiccounter ions are for example the ammonium ion, the potassium ion, thecalcium ion, the magnesium ion and, more preferably, the sodium ion.Suitable cations of organic bases are, among others, those of primary,secondary or tertiary amines, such as, for example, ethanolamine,diethanolamine, morpholine, glucamine, N,N-dimethylglucamine,tris(hydroxymethyl)aminomethane and especially N-methylglucamine.Suitable cations of amino-acids are, for example, those of lysine,arginine and omithine as well as the amides of any other acidic orneutral amino-acid such as for example lysine methylamide, glycineethylamide or serine methylamide.

According to a third aspect of the present invention, theabove-described substituted bis-indole derivatives of formula (I) andtheir metal complexes may be used in vitro, in vivo and/or ex vivo, forinstance in the form of their pharmaceutically acceptable salts and/orin the form of pharmaceutical compositions comprising them in admixturewith at least one pharmaceutically acceptable carrier, as diagnosticagents and/or therapeutic agents. For instance, these active ingredientsare useful for the manufacture of medicaments suitable for imaging orimaging-aided applications, including magnetic resonance imaging (MRI),nuclear scintigraphy (NS), MRI-aided applications or NS-aidedapplications or for the manufacture of imaging agents or imaging-aidedagents for use in such applications. This includes their use as in vivoeffective contrast agents, including multipurpose contrast agents, forvisualizing and/or identifying organs, parts of organs or systems suchas for example the vasculatory system, the hepatobiliary system or therenal-urinary system, tissues such as for example necrotic tissue, andfor visualizing and/or identifying diseases and pathologies. Diseasesinvolved in this aspect of the invention include ischemic insults suchas myocardial or cerebral infarction and space-occupying lesions (e.g.tumors or inflammatory lesions) in solid organs such as the liver,kidney, spleen, adrenal gland, etc. These agents are also useful in thefollow-up of a therapy, for instance the evolution of necrosis. Inparticular, these contrast agents are useful in medical applicationsinvolving necrosis and necrosis-related pathologies, such aspathological or therapeutic necrosis caused by pathologic ortherapeutically-induced ischemia or originating from trauma, radiationand/or chemicals, including therapeutic ablation, radiotherapy and/orchemotheraphy, myocardial and cerebral infarctions. For this purpose,they are administered to the human body, preferably enterally orparenterally, as therapheutic and/or diagnostic agents.

Pharmaceutically acceptable carriers for use in admixture with thebiologically-active ingredients of this invention are well known in theart of pharmacy and will be selected depending on the mode ofadministration to the patient (i.e. the mammal, in particular humans)involved. Most often, a suitable formulation is a physiologicallyacceptable liquid formulation, preferably an aqueous solution or anemulsion or suspension including conventional surfactants such aspolyethylene glycol.

In an another embodiment, the invention relates to a method forgenerating an image of at least a part of a body of a mammal, comprisingsystemically or locally administering to the mammal a contrast agenteffective amount of a metal-complexable substituted bis-indolederivative or a metal complex thereof having the formula (I).Preferably, the contrast agents of the invention are used systemicallyas diagnostic agents by parenteral administration, including intravenousinjection, at low doses, i.e. when a complexing metal such as gadoliniumis used, i.e. at doses from about 10 to about 500 μmoles gadolinium perkg body weight, preferably at doses ranging from about 10 to about 50μmoles gadolinium per kg body weight, the lower part of such rangesbeing still in vivo effective in case of systemic applications.

Alternatively, the contrast agents of the invention are also useful forlocal administration, e.g. including intracoronary administration in thecase of a patient with myocardial infarction. Depending on the specificcase, an effective local dose of the contrast agent of the invention maybe from 1 to about 5 μmoles gadolinium per kg body weight of the patientto be treated.

Yet alternatively, when a radioactive complexing metal such asindium-111 is used, the metal complex may be administered with aradioactivity in the range of about 20 to 200 MBq (megabecquerels). Whena radioactive complexing metal such as technetium-99 is used, the metalcomplex may be administered with a radioactivity in the range of about350 to 1,000 MBq.

Hereinbelow, the present invention is further described and explained byway of examples which are specific embodiments of the present inventionand should not be construed as limiting its scope.

EXAMPLE 1 Preparation of DTPA Monoethyl Monobenzylester

200 mmole of diethylene triamine pentaacetic acid (DTPA) bis anhydride(71.4 g) commercially available from Aldrich and 40 mmole drytriethylamine (TEA) in 1100 ml dry dimethylformamide were brought intosolution at 50° C. To the warm solution were added 200 mmole ethanol and200 mmole dry benzyl alcohol. After 2 hours reaction, solvents wereremoved under reduced pressure. The DTPA monoethyl monobenzylester wasseparated from the diethyl ester and the dibenzyl ester by preparativelow pressure reversed phase liquid chromatography. Therefore, theresidue was dissolved in 250 mM phosphate buffer (H₃PO₄/TEA at pH 6.5)and applied to a C18 silica column which was eluted with an acetonitrilegradient. High pressure liquid chromatography (210 nm monitoring,polymer column 4.6 mm×250mm, using a 25 mM phosphate/TEA buffer pH6.5—acetonitrile gradient, 1 ml/min) showed that pure DTPA monoethylmonobenzyl ester eluted from the preparative column with 7.5%acetonitrile/buffer (25 mM H₃PO₄/TEA at pH 6.5) to 15%acetonitrile/water. Pure fractions were pooled and concentrated underreduced pressure. Subsequent desalting of the preparation wasaccomplished by preparative C18 column chromatography (elution withacetonitrile/water). Solvent from the desalted material was removedunder reduced pressure. After removing excess water azeotropically withacetonitrile, the product was dried under vacuum over P₂O₅.

EXAMPLE 2 Preparation of DTPA Monoethyl Ester

DTPA monoethyl ester was prepared by hydrogenolysis of the purifiedcompound obtained in example 1 by dissolving it in 350 ml of a 70%ethanol/30% water mixture (volume/volume), then adding 2 g palladium onactivated carbon (Pd 10%). After 5 hours of a hydrogen gas treatmentunder a 20 p.s.i pressure, charcoal was removed by filtration over athin path of Celite. The residue was then washed with the same warmethanol/water mixture. Solvents from the combined filtrate and washingswere removed under reduced pressure. Drying in vacuum over P₂O₅ afforded27 g of DTPA monoethyl ester.

EXAMPLE 3 Preparation of DTPA Monoethyl Ester Mono Anhydride

The product of example 2 was converted to its mono anhydride derivativeby means of the acetic anhydride/pyridine method, making use of 250 mlacetic anhydride and 42 ml pyridine under a nitrogen gas atmosphere.Finally, 14 g (34.7 mmole) of NMR-1H characterized (dimethylsulfoxideD6) DTPA monoethylester monoanhydride was obtained as a white powder.

EXAMPLE 4 Preparation of3′-{[(2-alkyloxycarbonyl)-1H-indol-3-yl]-phenyl-methyl}-1H-indole-2-carboxylicacidester

The title compound was prepared from commercially available benzaldehydeand indole-2-carboxylic acid in a 90% yield, following the proceduredisclosed by Gränacher et al. in Helv. Chim. Acta (1924) 7:579–586.

EXAMPLE 5 Preparation of(3′-{[(2-hydrazinocarbonyl)-1H-indol-3-yl]-phenyl-methyl}-1H-indole-2-carbonyl)-hydrazine

5 g (10.7 mmole) of the intermediate obtained in example 4 and 10.73 ghydrazine monohydrate are dissolved in a mixture of 60 ml pyridine and30 ml methanol. After refluxing the mixture at 80° C. over night,solvents are removed under reduced pressure. The residue is treated byadding H₂O, H₂O/methanol and then acetonitrile; after each addition, thesolvent is removed under reduced pressure. Finally, the hydrazide isprecipitated from dichloromethane by the addition of acetonitrile, theprecipitate is collected by filtration and dried over P₂O₅, yielding 3.4g of the desired product. Identification thereof was confirmed by ¹H-and ¹³C-NMR spectroscopy. Spectra are recorded on a Gemini 200 MHzspectrometer available from Varian (Palo Alto, Calif.). Chemical shiftsare reported in ppm relative to tetramethylsilane (δ=0) as follows:¹H-NMR (DMSO) δ 4.51 (br s, NHNH2, 4H), 6.57–6.68 (m, ArH3, ArH4, 4H),6.99–7.11 (m, ArH2, ArH, 4H), 7.21 (m, ArH, 3H), 7.27 (s, CH, 1H), 7.40(d, ArH1, 2H), 9.62 (s, CONH, 2H), 11.40.

EXAMPLE 6 Preparation of the Bis-DTPA Amide of the Compound of Example 5(First Method)

3 g (6.85 mmole) of the compound of example 5 is dissolved in 200 ml dryDMF containing 12 ml NN-diisopropylethyl amine. The monoanhydride ofexample 3 (6.6 g, 16.4 mmole) is added and the mixture is stirred untilall starting hydrazide has disappeared. Completion of the reaction ismonitored by reversed phase high performance liquid chromatography(HPLC) (gradient elution of a C8/5 μm column using 25 mM TRIS/HCl bufferpH 7.4 containing 0.5 mM EDTA and acetonitrile). Solvents are removedunder reduced pressure. Protective ethyl ester groups are removed bydissolving the residue in diluted NaOH. A pH of 13.5 is maintained tillall ethyl esters are hydrolyzed. After alkaline hydrolysis, the pH isadjusted to 7.0 and the sample is applied to a preparative C18 silicacolumn. Low-pressure liquid chromatography (using a water-methanolgradient) affords the pure title compound (having a purity above 98% asdetermined by HPLC). After removal of solvents and drying over P₂O₅ apure white solid (4.3 g) is obtained in an overall 50% yield.Identification of the compound was performed as described in thefollowing example.

EXAMPLE 7 Prenaration of the Bis-DTPA Amide of the Compound of Example 5(Second Method)

To a solution of DTPA-bisanhydride (21.5 g, 60 mmole) and triethylamine(35 ml) in 200 ml of dry dimethylformamide (DMF) was slowly added 1 mlof water (55 mmole) in 50 ml of dry DMF over a period of 2 hours. 20mmole of the compound obtained in example 5 as then added and thereaction mixture was stirred overnight. After evaporation to dryness,the residue was dissolved at pH 8 (NaHCO₃ and 5N NaOH) and againevaporated to dryness to remove excess triethylamine. After dissolvingthe residue in 250 ml of 0.25M phosphate buffer pH 6.5 containing 2.5%of methanol, this solution was applied on a preparative C 18 column (1kg) in the same buffer. The column was eluted with a decreasing gradientof buffer and increasing amounts of acetonitrile. Pure fractions werecombined and evaporated to dryness, yield 75 g (containing phosphatesalts). The product obtained was dissolved in 150 ml water and desaltedon the same C 18 column in 2.5% of methanol, using a gradient ofacetonitrile, yielding 28 g (44%) of the desired product. Itsidentification is confirmed by the following spectra data: ¹H-NMR (D₂O)3.0–3.8 (methylene hydrogens of DTPA, 18H); 6.8–7.6 (aromatic hydrogensand —CH, 14H). Identification was further confirmed by mass spectrometryon a Micromass LCT instrument (time of flight machine) with electrosprayionisation detection, yielding a peak at 579 (half of molecular mass,corresponding to a bisanionic compound at pH 7).

EXAMPLE 8 Preparation of the Bis Gadolinium Complex of the Compound ofExample 6

Gadolinium acetate was incrementally added to an aqueous solution of thecompound of example 6. After each addition the pH was adjusted to 7.4 bymeans of NaOH (1.0 M). Formation of mono- and bis-gadolinium chelateswas monitored by HPLC, knowing that chelation increases retention time.Addition of gadolinium acetate was stopped when the compound wasvirtually completely converted into the bis- and mono-gadoliniumcomplexes, the latter amounting to minor amounts (DTPA moieties notinvolved in gadolinium complexation was less than 10%). Identity of thecomplex was confirmed by mass spectrometry on a Micromass LCT instrument(time of flight) with electrospray ionisation detection. Peaks arepresent at 1497 dalton (i.e. the molecular mass of the compound withsingle negative charge+Na) and at 748 dalton (i.e. half of thatmolecular mass, corresponding to the bis-anionic compound). Both peaksshow the characteristic distribution of the different stable isotopes ofgadolinium.

EXAMPLE 9 Preparation of3′-{[(2-alkyloxycarbonyl)-1H-indol-3-yl]-methylene}-1H-indole-2-carboxylicacid ethyl ester.

The title compound was prepared from formaldehyde andindole-2-carboxylic acid in a 28% yield, following the proceduredisclosed by Gränacher et al. in Helv. Chim. Acta (1924) 7:579–586.

EXAMPLE 10 Preparation of(3′-{[(2hydrazinocarbonyl)-1H-indol-3-yl]-methylene}-1H-indole-2-carbonyl)-hydrazine

7.35 g (20 mmole) of the intermediate obtained in example 10 and 30 mlhydrazine monohydrate are dissolved in a mixture of 100 ml pyridine and30 ml methanol. After refluxing the mixture at 80° C. over night,solvents are removed under reduced pressure. The residue is treated byadding H₂O, H₂O/methanol then acetonitrile; after each addition, thesolvent is removed under reduced pressure. Finally, the hydrazide isprecipitated from dichloromethane by the addition of acetonitrile, theprecipitate is collected by filtration and dried over P₂O₅, yielding 6.5g of the desired product. Its identification is confirmed by thefollowing spectra data: ¹H-NMR (DMSO) δ 4.63 (br s, NHNH2, 4H), 4.92 (s,CH2, 2H), 6.81 (t, ArH2, 2H), 7.09 (t, ArH3, 2H), 7.33 (d, ArH4, 2H),7.43 (d, ArH1, 2H), 9.52 (s, CONH, 2H), 11.12 (s, NH, 2H).

EXAMPLE 11 Preparation of[{2-[N′-(3-{[2-(N′-{2-[(2-{[2-bis(carboxymethyl-amino)-ethyl]carboxymethylsmino}-ethyl)carboxymethyl-amino]-acetyl}-hydrazinocarbonyl)-1H-indol-3-yl]-methylene}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-aceticacid sodium salt

Preparation was effected as described in example 6 but starting from6.33 g of the compound of example 10, yielding the desired sodium saltin a 70% yield. Its identity was confirmed by mass spectrometry on aMicromass LCT instrument (time of flight machine) with electrosprayionisation detection, yielding a peak at 541 dalton, corresponding tothe bisanionic compound at pH 7.

EXAMPLE 12 Preparation of the Bis Gadolinium Complex of[{2-[N′-(3-[{2-(N′-{2-[(2-{[2-bis(carboxymethyl-amino)-ethyl]carboxymethylamino}-ethyl)carboxymethyl-amino]-acetyl}-hydrazinocarbonyl)-1H-indol-3-yl]-methylene}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-aceticacid sodium salt.

The compound of example 11 (12.0 g, 10 mmole) was dissolved in 200 mlwater; a solution of 11.5 mmole of gadolinium acetate Gd(0Ac)₃.3H₂O in100 ml of water was then slowly added while maintaining the pH at 7 with25% NH₃. The Gd-complex was evaporated to dryness, washed withacetonitrile and dried under vacuum over phosphorus pentoxide and sodiumhydroxide for removing ammonium acetate. 13.25 g (90%) of the titlecomplex was obtained. Its identity was confirmed by mass spectrometry ona Micromass LCT instrument (time of flight machine) with electrosprayionisation detection, yielding peaks at 711 dalton (corresponding tohalf of the molecular mass (bisanionic compound+Na)) and at 474,corresponding to ⅓ of that molecular mass for the tris-anionic compound.Both peaks show the characteristic distribution of the different stableisotopes of gadolinium.

EXAMPLE 13 Preparation of{4,7-Bis-carboxymethyl-10-[({3-[(2-{N′-[(4,7,10-tris-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetyl]-hydrazinocarbonyl}-1H-indol-3-yl)-phenyl-methyl]-1H-indole-2-carbonyl}-hydrazino)-2-oxo-ethyl]-1,4,7,10-tetraaza-cyclododec-1-yl}-aceticacid

1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA, 8.08 g, 20 mmole)was dissolved in a mixture of 15 ml of ammonia and 250 ml of dry DMSO bystirring and sonicating.O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU, 9.63 g, 30 mmole) was added and allowed to react for 30 minutes.The compound of example 5 (2.63 g, 6 mmole) was then added and allowedto react for 60 minutes. High performance liquid chromatography (HPLC)on a RP-18 Chromolith column Merck (10 cm length) eluted with gradientmixtures of acetonitrile in 0.05 M ammonium phosphate buffer pH 6.8 (0to 80% acetonitrile over a period of 30 minutes) showed that thereaction mixture contained a mixture of the mono- and bis-DOTAderivatives. Isolation of the bis-DOTA compound was carried out on a1-kg RP-18 column, using as the eluent 10% ammonium acetate in 2.5%methanol, followed by 5% methanol and finally 10% methanol. The titlecompound was isolated from the appropriate fractions in a 40% yield(3.11 g). Its identity was confirmed by mass spectrometry on a MicromassLCT instrument (time of flight machine) with electrospray ionisationdetection, yielding a peak at 616 dalton (corresponding to half of themolecular mass (bis-anionic compound+Na).

EXAMPLE 14 Preparation of3,3′-p-methoxybenzylidenebis(indole-2-carboxylic acid ethyl ester) or3′-{[(2-alkyloxycarbonyl)-1H-indol-3-yl]-(4-methoxyphenyl)-methyl}-1H-indole-2-carboxylicacid ethyl ester

The title compound was prepared, from 4-methoxybenzaldehyde andindole-2-carboxylic acid in a 85% yield, following the proceduredisclosed by Gränacher et al. in Helv. Chim. Acta (1924) 7:579–586.

EXAMPLE 15 Preparation of(3′-{[(2-hydrazinocarbonyl)-1H-indol-3-yl]-(4-methoxyphenyl)-methyl}-1H-indole-2-carbonyl)-hydrazine

Preparation was effected, starting from the compound of example 15, in a80% yield, by using the procedure described in detail for example 5.Identification of the resulting compound is confirmed by the followingspectra data: ¹H-NMR (DMSO) δ 3.72 (s, OCH3, 3H), 4.53 (br s, NHNH2,4H), 6.66 (m, ArH4, ArH3, 4H), 6.80 (d, ArH5, 2H), 6.91 (d, ArH6, 2H),7.03–7.12 (m, ArH2, 2H), 7.18 (s, CH, 1H), 7.40 (d, ArH1, 2H), 9.59 (s,CONH, 2H), 11.39 (s, NH, 2H).

EXAMPLE 16 Preparation of[{2-[N′-(3-{[2-(N′-{2-[(2-{[2-bis(carboxymethyl-amino)-ethyl]carboxymethylamino}-ethyl)carboxymethyl-amino]-acetyl}-hydrazinocarbonyl)-1H-indol-3-yl]-(4-methoxyphenyl)-methyl}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-aceticacid sodium salt.

Preparation starting from the compound of example 15 was effectedessentially as described in example 6 above, providing the titlecompound in a 53% yield. The identity of the resulting compound wasconfirmed by the following spectra data: ¹H-NMR (DMSO-D20) δ 3.1–3.8 (m,36H, CH2), 3.68 (s, OCH3, 3H), 6.6–6.8 (m, ArH,6H), 6.99 (s, CH, 1H),7.1–7.2 (m, 4H), 7.53 (d, ArH, 2H). Identity of the compound was furtherconfirmed by mass spectrometry on a Micromass LCT instrument (time offlight machine) with electrospray ionisation detection, yielding a peakat 595 (half of molecular mass, corresponding to a bisanionic compoundat pH 7).

EXAMPLE 17 Preparation of the bis gadolinium Complex of[{2-[N′-(3-{[2-(N′-{2-[(2-{[2-bis(carboxymethyl-amino)-ethyl]carboxymethylamino}-ethyl)carboxymethyl-amino]-acetyl}-hydrazinocarbonyl)-1H-indol-3-yl]-(4-methoxyphenyl)-methyl}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-aceticacid sodium salt.

The compound of example 16 was quantitatively converted to itsbis-gadolinium complex by following essentially the same procedure asdescribed in example 8. Identity of the complex was confirmed by massspectrometry on a Micromass LCT instrument (time of flight) withelectrospray ionisation detection. Peaks are present at 763 dalton (halfof the molecular mass, corresponding to the bis-anionic compound) and508 dalton (one third of the molecular mass: compound with threenegative charges). Both peaks show the characteristic distribution ofthe different stable isotopes of gadolinium.

EXAMPLE 18 Preparation of the indium-111 complex of[{2-[N′-(3-{[2-(N′-{2-[(2-{[2-bis(carboxymethyl-amino)-ethyl]carboxymethylamino}-ethyl)carboxymethyl-amino]-acetyl}-hydrazinocarbonyl)-1H-indol-3-yl]-(4-methoxyphenyl)-methyl}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-aceticacid sodium salt

In a 10-ml vial, 2.5 mg of the compound of example 6 is dissolved in 0.2ml of a 0.1 molar citrate buffer pH 4.0. To this solution is added 0.4ml of a [¹¹¹In] indium chloride solution (Tyco Healthcare, DRN 4901)containing 26 megabecquerel (MBq) of ¹¹¹In. The solution is incubatedfor 30 minutes at 23° C. and then analysed by HPLC on an X-Terra RP18column (250 mm×4,6 mm, particle size 5 μm), eluted with gradual mixturesof 0.1 molar ammonium acetate and acetonitrile (0% acetonitrile atstart, linearly increased to 90% acetonitrile after 30 minutes). Theeluate is monitored for radioactivity using a NaI(TI) scintillationdetector and for UV absorbance using a UV-detector. The only peak ofradioactivity elutes at 19.5 minutes, immediately after the UV-peakcorresponding to the compound of example 6.

EXAMPLE 19 In vivo Test of Rat Liver Infarction

Adult Wistar rats (weighing between 300 and 400 g) were anesthetizedwith intraperitoneal injection of pentobarbital (Nembutal® availablefrom Sanofi Santé Animale, 1130 Brussels, Belgium) at a dose of 40mg/kg. Under laparotomy, reperfused hepatic infarction was induced bytemporarily clamping the hilum of the right liver lobe for 3 hours.After reperfusion by declamping hepatic inflow, the abdominal cavity wasclosed with 2-layer sutures and the rats were left to recover for 8 to24 hours after surgery.

Animals were anesthetized again as above for magnetic resonance imaging(MRI). The tail vein of the rat was cannulated with a G27 infusion set(available from Vicon, Belgium) connecting to a 1 ml tuberculin syringeloaded with a 43 mM Gd solution of the complex of example 8. The rat wasimaged at 1.5 T scanner (Magnetom Vision, available from Siemens,Erlangen, Germany) within a cylindrical copper coil. T2-weighted(repetition time/echo time TR/TE=3000 ms/90 ms) and T1-weighted(TR/TE=420 ms/12 ms) spin echo sequences were applied. Other MRIparameters were as follows: slice thickness was 2 mm (without gap); thefield of view was 7.5 cm×10 cm, with a matrix of 192×256. Twoacquisitions were averaged, resulting in a measurement time of about 3minutes. A glass tube containing 0.02% CuSO₄ solution was placed besidethe rat as an external standard for normalization of signal intensity(SI) values. Together with a precontrast T1-w imaging, only one T2-wmeasurement was performed at the beginning to verify the presence ofinfarcted liver lobe. Rats were scanned on transverse sections beforeand after contrast injection of the compound of example 8 at aconcentration of 0.05 mmole Gd per kg body weight. Postcontrast T1-w MRIwas effected 5 minutes (early phase), 40 minutes and 24 hours (latephase) thereafter.

At the end of imaging studies, rats were sacrificed by an intravenousoverdose of phenobarbital and placed in a deep freezer (below −20° C.)overnight in the same position as that during MR imaging. The frozenrats were sectioned in the transverse plane similar to that on MRI inorder to match the imaging and histologic findings. Another approach wasto perfuse freshly excercised liver (or other organs) with a2,3,5-triphenylterazolium solution in order to provoke staining ofviable tissue.

For the purpose of microscopy, tissue samples were fixated, sectioned,stained and analyzed according to standard procedures.

Results of MRI-imaging and post mortem macroscopic analysis as shown inFIG. 3 (A-E) demonstrate the blood pool effects of the compound ofexample 8 and its ability to visualize necrosis. Before contrast, theinfarcted liver lobe (arrow) is almost isointense relative to the normalliver (A). While immediately after (B) injection of the compound, thesignal intensity of normal liver is enhanced and the infarcted lobe(arrow) remains hypointense. All intrahepatic vessels exhibit strongsignal intensity, a feature characterizing the reperfused infarctionmodel. 40 minutes later (C), the contrast between normal and infarctedliver is reversed due to a combination of events comprising (1) gradualcontrast perfusion and diffusion into the necrotic lobe, (2) decliningplasma concentrations (due to slow clearance fom the blood andelimination from the body) and (3) retention of the contrast agent inthe necrosis by biospecific binding to necrotic tissue or componentsthereof. The simultaneous occurrence of these dynamic events causeheterogeneous contrast enhancement of necrosis in the early phase. 24hours after administration (D), the signal intensity of both the normalliver and the vessels have largely decreased. However the infarcted lobedisplays a persistent homogenous contrast enhancement, illustrating thatthe complex of example 8 exhibits necrosis-specificity. Comparison ofthe in vivo MR-image of FIG. 3D with post mortem histologic localisationof necrosis (FIG. 3E showing the necrotic tissue of a rat sectioncorresponding to the MRI-slice of FIG. 3D), confirms that the observedlate phase contrast enhancement exactly matches necrosis. Hence, thecompound of example 8 has necrosis seeking ability and is useful forunambiguous identification and in vivo visualization of necrosis.

In addition, blood clearance of the compound of example 8 was slow (T1/2is about 2.5 hours). In conjunction with its ability to reversibly bindto serum proteins, in particular to serum albumin, this means that italso exhibits bloodpool effects.

EXAMPLE 20 In Vivo Test of Rat Liver Infarction at Low Dose

The complex of example 8 (used in an appropriate dilution, by means ofsaline buffer, of a preparation of 350 mM Gd) was further tested in thesame rat model of reperfused liver infarction as described in example 19at both normal and low dose. One group of rats received a dose of about50 μmole gadolinium per kg body weight (as in example 19) while theother group received a dose of about 10 μmole gadolinium per kg bodyweight. MRI-images and histologic correlation, performed under the sameconditions as in example 19, demonstrated unambiguous enhancement ofnecrotic tissue even at a dose as low as 10 μmole Gd/kg. 24 hours afterinjection of the complex, the observed ratio between normal and necroticliver was equal to or higher than 1.3 in rats who received the 10 μmoleGd/kg dose. In rats who received the 50 μmole Gd/kg dose, this ratio wasgreater than 1.6. Taking into account that the agent is cleared ratherslowly and that normal liver is involved in eliminating the complex fromthe body, and that 24 hours after injection there is still enhancementin normal liver on MRI images, therefore when infarction is located inthe myocardium or the brain, or more generally in organs and/or tissuesnot involved in elimination of the complex, the contrast ratio will bestill much higher. Accordingly, doses below 15 μmole Gd/kg will beeffective in vivo when the complex is used in systemic applications.When the site of necrosis permits local administration of the agent(such as for example intracoronary administration in the case ofmyocardial infarction), then doses far below those effective in systemicapplications can be used.

EXAMPLE 21 In Vivo Test of Rat Liver Infarction with DelayedAdministration

The compound of example 8 was further tested in the same rat model ofreperfused liver infarction as described in example 8 at normal dose(about 50 μmole Gd/kg) and low dose (about 15 μmole Gd/kg), butadministering the said compound after a delay of 48 hours followinginducing the liver infarction. MRI-images and histologic correlation,performed under the same conditions as in example 19, revealed that evenunder such conditions, excellent enhancement of necrosis was obtained,i.e. the observed contrast ratios were similar as in examples 19 and 20.

EXAMPLE 22 Toxicity of a Gadolinium Complex

Possible toxic side effects of the complex of example 8 were studied infive normal Wistar rats that received a bolus intravenous injection ofabout 1,000 μmole Gd/kg. After treatment, rats were kept in standardconditions, having free access to water and food. Being carefullymonitored during the first 10 hours after injection, they did not showany sign of abnormal behaviour. After 24 hours, the rats weresacrificed, organs were excised and macroscopically inspected. Tissuesamples were collected and prepared for microscopic analysis. Postmortem inspection of organs did not reveal any abnormalities. Extensivemicroscopic analysis of tissue samples (liver, heart, kidney, brain andmuscle) did not show any abnormal histology and did not reveal anyhistological indication of toxicity (such as e.g. inflamation, necrosisand the like).

EXAMPLE 23 In Vivo Test of Pig Myocardial Infarction

Pigs weighing about 40 kg were sedated with intramuscular xylazine(commercially available under the tradename Rompun from Bayer) at 2.5ml/kg, anesthetized with an intravenous bolus of 60 mg of sodiumpentobarbital (Nembutal, Sanofi), intubated and ventilated on a positivepressure ventilator. Anesthesia was maintained by dosed infusion ofsodium pentobarbital beneath the level of spontaneous respiration.Myocardial infarction was induced through a surgical procedure. Afterleft posterolateral thoracotomy and opening of the pericardium, the leftcoronary artery was identified and a loose snare loop with a 3–0 silksuture was applied around the artery and lead out of the chest through asmall incision. The chest was closed after evacuation of thepneumothorax. After baseline imaging, obstructive myocardial infarctionwas induced by tightening of the snare through a 7 French latex tube for150 to 180 minutes. The ischemic effect of the procedure was verified byelectrocardiography (hereinafter referred as ECG) changes. Coronaryperfusion was restored by subsequent removal of the occluder.

30 to 60 minutes after reperfusion, a dose of 0.05 mmole/kg of thecompound of example 8 was administered intravenously. Cardiac MRI wasperformed one, two, four, six and twelve hours respectively afteradministration on a 1.5 T clinical imager (Siemens Magnetom Vision,Erlangen, Germany) with gradient switching capabilities of 25 mT/m in300 μsec. All pigs were positioned in the supine position in thestandard surface coil, centered on the xyphoid process of the sternum.MR images were obtained in the true short axis and optionally in thelong-axis. Under ventilator-assisted breath-holding, we used anECG-triggered and segmented single-slice turboFLASH sequence. Thesequence starts by applying a 180° inversion pulse for annulling thesignal from the cardiac cavity, after which an echo train of 33 echoesis acquired after each R-peak of the ECG. With a matrix of 165×256, thisresults in filling of k-space in 5 heartbeats. Other sequence parameterswere: TR/TE/α: 7.5 msec/4.3 msec/25°, inversion of 600 ms. The field ofview was 240×320 mm and the slice thickness 6 mm.

At the end of imaging study, the animals were sacrificed and the excisedheart was stained in a solution of buffered triphenyltetrazoliumchloride (hereinafter referred as TTC). TTC staining results in redcoloration of non-infarcted tissue, whereas necrotic areas exhibit apale color. All slices were photographed and digitized for morphometry.

Results of MRI-imaging and post mortem macroscopic analysis as shown inFIGS. 4(A-D) demonstrate the effects of the compound of example 8.Before contrast, the infarct (arrow) appeared isointense and invisible(A). Respectively 2 hours (B) and 6 hours (C) after intravenousinjection of the compound of example 8 at a dose of 0.05 mmole/kg, thesignal intensity of infarct (arrow) was persistently enhanced. The paleinfarcted region (arrow) on TTC histochemically stained sectionconfirmed the above MRI finding (D).

EXAMPLE 24 (Comparative) and 25—Effect of in Vivo Intravenous Injectionin a Rabbit

For the purpose of a comparison with a known contrast agent, MRangiographic images of a rabbit were taken (shown in FIG. 5) afterintravenous injection of:

-   -   a commercial MRI contrast agent (Gd-DTPA commercially available        under the tradename Magnevisto® from Schering A G, Berlin,        Germany) at a concentration of 0.1 mmole per kg bodyweight        (A-C), and    -   the complex of example 8 at a concentration of 0.05 mmole per kg        bodyweight (A′-C′).

More specifically, immediately after Gd-DTPA injection, the abdominalaorta (arrow) was moderately enhanced by the first pass of the contrastagent (A). One minute after contrast, the aorta (arrow) could only befaintly enhanced (B). Six minutes after contrast, the aorta was onlonger enhanced as a result of complete washout of the agent from theblood circulation (C). By contrast, during the first pass immediatelyafter intravenous injection, the complex of example 8 caused a strongercontrast enhancement of the abdominal aorta (arrow) (A′). Furthermore,the induced vascular contrast enhancement (arrow) persisted over 20minutes (B′) through 70 minutes (C′) after contrast.

EXAMPLE 26 In Vivo Tests in a Rat Model of Liver Metastasis

Under the same general anaesthesia procedure as in example 19, the liverof Wistar rats was implanted with a cube (1×1×1 mm³) of freshlyharvested R1 rhabdomyosarcoma tissue. One week after implantation, thetumor size grew to 0.8–1.2 cm in diameter, ready for radiofrequencyablation (RFA). The RFA protocol was as follows: after anaesthesia, anincision was made along the primary incision for tumor implantation.Under visual inspection, an 18 Gauge cool-tip electrode (Radionics,Burlington, Mass., USA) was directly inserted into the liver tumor.Radiofrequency current was delivered from a RF generator (RFG-3E,available from Radionics) under power control mode at 30W for 20–30seconds depending on the size of the tumor. The ablation volume coveredboth the entire tumor and a 3–5 mm rim of peritumoral liver parenchyma.After RFA, the electrode was withdrawn and the incision closed. Theefficacy of the therapy was evaluated with contrast enhanced MRI, usingthe compound of example 8 at a concentration of 0.05 mmole per kgbodyweight, and histopathology.

Results of MR images (A-E) and cross section (F) of a rat with liverimplantation of rhabdomyocarcoma treated with radiofrequency ablation,as shown in FIG. 6(A-F), demonstrate the effects of the compound ofexample 8. Before contrast, the treated tumor (arrow) appearedisointense and invisible (A). Respectively 1 minute (B) and 10 minutes(C) after intravenous injection of the compound of example 8 at a doseof 0.05 mmole per kg body weight, the signal intensity of liver ismoderately enhanced leaving the lesion (arrow) as a sphericalhypointense region. Respectively 6 hours (D) and 24 hours (E) aftercontrast, when the liver returned to its normal signal intensity, thelesion (arrow) including the ablated tumor and a layer of surroundingnecrotic liver tissue showed a striking rim contrast enhancement,suggesting a complete tumor eradication. The pale necrotic region(arrow) apparent on the histological section (F) confirmed the above MRIfinding.

EXAMPLE 27 Preparation of indole-3-carboxylic acid ethyl ester

The title compound is prepared starting from indole-3-carboxylic acid(commercially available from Aldrich) by esterification with ethanolusing standard methods of organic synthesis (e.g. J. March, AdvancedOrganic Chemistry, Reactions, Mechanisms and Structure (1992, 4^(th)edition) 393–394 (Wiley interscience, New York).

EXAMPLE 28 Preparation of2′-[(3carboxyethyl-1H-indole-2yl)-phenyl-methyl]-1H-indole-3-carboxylicacid ethyl ester

The title compound is prepared starting from benzaldehyde and from thecompound of example 27, using the method described in example 4.

EXAMPLE 29 Preparation of the bis-hydrazide of2′-[(3-carboxyethyl-1H-indole-2yl)-phenyl-methyl]-1H-indole-3-carboxylicacid

The title compound is prepared from the compound of example 28 using themethod described in example 5.

EXAMPLE 30 Preparation of the Bis DTPA Conjugate with the bis-hydrazideof 2′-[(3-carboxy-1H-indole-2yl)-phenyl-methyl]-1H-indole-3-carboxylicacid

The title compound is prepared from the compound of example 29 using themethod described in example 6.

EXAMPLE 31 Preparation of the bis-gadolinium Complex of[{3-[N′-(2-{[3-(N′-{2-[(2-{[2-bis(carboxymethyl-amino)-ethyl]carboxymethylamino}-ethyl)carboxymethyl-amino]-acetyl}-hydrazino-carbonyl)-1H-indol-2-yl]-phenyl-methyl}-1H-indole-3carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-aceticacid sodium salt.

The title compound is prepared from the compound of example 30 using hemethod described in example 8.

EXAMPLE 32 Preparation of2′-[2-(3-carboxyethyl-1H-indole-2-yl)-ethylene]-1H-indole-3′-carboxylicacid ethyl ester

The title compound is prepared starting from2′-[2-(3-carboxy-1H-indole-2-yl)-ethyl]-1H-indole-3′-carboxylic acid(the latter being prepared following the procedure described by D.Nagarathnanet al. in Ind. J. Chem. (1981) 20B, 796–797, byesterification with ethanol using standard methods of organic synthesis(e.g. J. March cited supra).

EXAMPLE 33 Preparation of the dihydrazide of2′-[2-(3-carboxyethyl-1H-indole-2-yl)-ethylene]-1H-indole-3′-carboxylicacid

The title compound is prepared from the compound of example 32 using themethod described in example 5.

EXAMPLE 34 Preparation of the bis-DTPA Conjugate of the dihydrazide of2′-[2-(3-carboxyethyl-1H-indole-2-yl)-ethylene]-1H-indole-3′-carboxylicacid

The title compound is prepared from the compound of example 33 using themethod described in example 6.

EXAMPLE 35 Preparation of the bis-gadolinium Complex of the bis-DTPAConjugate of the dihydrazide of2′-[2-(3-carboxyethyl-1H-indole-2-yl)-ethylenel-1H-indole-3′-carboxylicacid

The title compound is prepared from the compound of example 34 using themethod described in example 8.

EXAMPLE 36 Preparation of the bis-hydrazide of3′-(2-carboxy-1H-indole-3-ylsulfanyl)-1H-indole-2′-carboxylic acid

The title compound is prepared starting from3′-(2-carboxyethyl-1H-indole-3-ylsulfanyl)-1H-indole-2′-carboxylic acidethyl ester (the latter being prepared following the procedure describedby J. Szmuszkovicsz, J. Org. Chem. (1964) 29: 178–184), byesterification with ethanol using standard methods of organic synthesis(e.g. J. March cited supra)

EXAMPLE 37 Preparation of the bis-DTPA Conjugate of the bis-hydrazide of3′-(2-carboxy-1H-indole-3-ylsulfanyl)-1H-indole-2′-carboxylic acid

The title compound is prepared from the compound of example 36 using themethod described in example 6.

EXAMPLE 38 Preparation of the bis-gadolinium Complex of the bis-DTPAConjugate of the bis-hydrazide of3′-(2-carboxy-1H-indole-3-ylsulfanyl)-1H-indole-2′-carboxylic acid

The title compound is prepared from the compound of example 37 using themethod described in example 8.

EXAMPLE 39 Preparation of the bis-DTPA Conjugate of2′-(3-(2-aminoethyl)-1H-indole-2-ylsulfanyl)-1H-indole-3′-ethyl-2-amine

The title compound is prepared from dithio-2,2′-ditryptamine (the latterbeing obtained following the procedure described by Barbier et al. in J.Heterocycl. Chem. (1989) 26:265–267), using the method described inexample 6.

EXAMPLE 40 Preparation of the Bisgadolinium Complex of the bis-DTPAConjugate of2′-(3-(2-aminoethyl)-1H-indole-2-ylsulfanyl)-1H-indole-3′-ethyl-2-amine

The title compound is prepared from the compound of example 39 using themethod described in example 8.

EXAMPLE 41 Preparation of2′-(3-(2-ethylcarboxyethyl)-1H-indole-2-ylsulfanyl)-1H-indole-3′-propionicacid ethyl ester

The title compound is prepared starting from3′-[2-(3-(2-carboxyethyl)-1H-indole-2-ylsulfanyl)-1H-indol-3′-yl]-propionic_acid(the latter being obtained following the procedure disclosed by Thompsonet al., J. Med. Chem. (1994) 37(5):598–609 and U.S. Pat. No. 5,464,861)by esterification with ethanol using standard methods of organicsynthesis (e.g. J. March cited supra)

EXAMPLE 42 Preparation of the bis-hydrazide of3′-[2-(3-(2-ethylcarboxyethyl)-1H-indole-2-ylsulfanyl)-1H-indol-3′-yl]-propionicacid

The title compound is prepared from the compound of example 41 using themethod described in example 5.

EXAMPLE 43 Preparation of the bis-DTPA Conjugate of the bis-hydrazide of3′-[2-(3-(2-ethylcarboxyethyl)-1H-indole-2-ylsulfanyl)-1H-indol-3′yl-]-propionicacid

The title compound is prepared from the compound of example 42 using themethod described in example 6.

EXAMPLE 44 Preparation of the bis-gadolinium Complex of the bis-DTPAConjugate of the bis-hydrazide of2′-(3-(2-ethylcarboxy)-1H-indole-2-ylsulfanyl)-1H-indole-3′-propionicacid

The title compound is prepared from the compound of example 43 using themethod described in example 8.

1. A substituted bis-indole derivatives of formula (I) or a mentalcomplex, wherein said metal complex consisting of bis-indole derivativesof formula (I) and a radioactive or non-radioactive metal ion of anelement with an atomic number selected from the group consisting of 21to 32, 37 to 39, 42 to 44, 49, 50 or 57 to 83, and wherein the formula(I) is hereunder:

a pharmaceutically acceptable salt thereof wherein: L represents asingle bond or an optionally substituted linking agent (Li) whichcovalently links together the carbon atoms being respectively inpositions 2 or 3 and 2′ or 3′ on the heterocyclic indole rings, (Li)being an optionally substituted alkylene group; R₁ and R₂ are optionalsubstituents of any free position of the phenyl rings of the indolylgroups and are each independently selected from halogen atoms, straightchain alkyl groups of 1 to 6 carbon atoms, branched chain alkyl groupsof 3 to 7 carbon atoms, cycloalkyl groups of 3 to 7 carbon atoms, alkoxygroups of 1 to 6 carbon atoms, nitro, trifluoromethyl, cyano, carboxylicacid, sulfonic acid, carboxylic acid esters wherein the ester groupderives from an alkyl group having 1 to 4 carbon atoms, substituted orunsubstituted carboxamides CO—NR₄R′₄ and substituted or unsubstitutedamines NR₄R′₄ wherein R₄ and R′₄ are each independently selected fromhydrogen and optionally substituted C₁–C₂₀ branched chain or straightchain alkyl groups or C₆–C₂₀ aryl groups or C₆–C₂₀ alkylaryl groups; R₃is an optional substituent of the linking agent (Li) and is selectedfrom optionally substituted aryl groups and optionally substitutedbranched chain or straight chain alkyl groups; q, p and r are integersindicating the number of the respective substituents R₁, R₂ and R₃ andare each independently selected from 0 to 4, provided that r is 0 when Lis a single bond; C₁ and C₂ are metal chelating agent-containingsubstituents of the heterocyclic rings of the indolyl groups; m and nare integers indicating the number of the respective metal chelatingagent-containing substituents C₁ and C₂ and are each 0 or 1, providedthat the sum of m and n is at least 1; and wherein R₁, R₂ or R₃independently does not represent a heterocycle or heteroaryl, andwherein R₁, R₂ or R₃ independently is not substituted with a heterocycleor heteroaryl.
 2. The bis-indole derivative or a metal complex accordingto claim 1, wherein m is 1 and n is
 1. 3. The bis-indole derivative orametal complex according to claim 1, wherein the substituent(s) on thearyl group R₃are independently selected from the group consisting ofhalogen atoms, saturated or unsaturated hydrocarbon groups having 1 to 4carbon atoms, alkoxy groups having 1 to 4 carbon atoms, cyano,carboxylic acid, sulfonic acid, carboxylic acid esters wherein the estergroup derives from an alkyl group having 1 to 4 carbon atoms,substituted or unsubstituted carboxamicies CO—NR₄R′₄ and substituted orunsubstituted amines NR₄R′₄ wherein R₄ and R′₄ are each independentlyselected from the group consisting of hydrogen, optionally substitutedC₁–C₂ branched chain or straight chain alkyl groups, C₆–C₂₀ aryl groupsand C₆–C₂₀ alkylaryl groups.
 4. The bis-indole derivative or a metalcomplex according to claim 1, wherein each of C₁ and C₂ is independentlyrepresented by the formula -(Sp)_(s)-CA, wherein (Sp) is a Spacingagent, s is an integer selected from 0 and 1, and CA is a chelatingagent.
 5. The bis-indole derivative or a metal complex according toclaim 1, wherein each of C₁ and C₂ is independently represented by theformula -(SP)_(s)-CA, wherein CA is a chelating agent wherein a is 1 andwherein (Sp) is a spacing agent represented by the formula—(X)_(t)-(Sp′)-, wherein (Sp′) is a molecule bearing at one end a firstfunctional group (i) suitable for reacting with a carboxylle acidfunction or with a primary or secondary amine and at the other end asecond functional group (ii) suitable for reacting with the chelatingagent CA, t is 0 or 1 and X is a carbonyl group or a secondary aminogroup.
 6. The bis-indole derivative or a metal complex according toclaim 1, wherein each of C₁ and C₂ is independently represented by theformula -(SP)_(s)-CA, wherein (Sp) is a spacing agent selected from thegroup consisting of bis-amino and amino-acids, s is an integer selectedfrom 0 and 1, and CA is a chelating agent.
 7. The bis-indole derivativeor a metal complex according to claim 1, wherein each of C₁ and C₂ isindependently represented by the formula -(Sp)_(s)-CA, wherein (Sp) is aspacing agent, s is an integer selected from 0 and 1, and CA is achelating agent comprising carboxymethyl groups —CH₂COOR₄ wherein R₄ isselected from the group consisting of hydrogen and optionallysubstituted C₁–C₂₀ branched chain or straight chain alkyl groups orC₆–C₂₀ aryl groups or C₅–C₂₀ alkylaryl groups.
 8. The bis-indolederivative or a metal complex according to claim 1, wherein each of C₁and C₂ is independently represented by the formula -(Sp)_(s)-CA, wherein(Sp) is a spacing agent, s is an integer selected from 0 and 1, and CAis a chelating agent comprising one or more moieties selected from thegroup consisting of bisamine-bisthiol, bisamine-bisoxime,monomercapto-triamide, diamide-dithiol, monoamine-monoamide-dithioltetramine, monoamine-diamide-monothiol monoamine-monothboether-dithiol,monoamine-monothiol, monoamide-diamine-monathiol and diphosphine basedmoieties.
 9. The bis-indole derivative or a metal complex according toclaim 1, wherein each of C₁ and C₂ is independently represented by theformula -(Sp)_(s)-CA, wherein (Sp) is a spacing agent, s is an integerselected from 0 and 1, and CA is a chelating agent selected from thegroup consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminopentaacetic acid (DTPA), trans-1,2-cyclohexanediaminetetraacetic acid (CDTA), 1,4,7,10-tetreaza-cyclododecane tetraaceticacid (DOTA), 1,4,7-triazacyclononanetriacetic acid,1,4,8,11-tetraazacyclotetra-decane tetraacetic acid (TETA),ethyleneglycol-O,O′-bis(2-aminoethyl)-tetraacetic acid (EGTA),N,N-bis(hydroxybenzyl)ethylene-diamine-N,N′-diacetic acid (HBED),triethylene-tetramine hexaacetic acid (TTHA), hydroxyethyldiaminetriacetic acid (HEDTA), 1,5,9-triazacyclo-dodecanetriacetic acid, andanalogues thereof.
 10. The bis-indole derivative or a metal complexaccording to claim 1, wherein each of C₁ and C₂ is independentlyrepresented by the formula -(SP)_(s)-CA, wherein (Sp) is a spacingagent, s is an integer selected from 0 and 1, and CA is a chelatingagent selected from the group consisting of mercaptoacetyl triglycine,hexamethylpropylene diamine dioxime, ethylene dicysteine, ethylenecysteine cysteamine, cysteinylglycine cysteine,bismercaptoacetyl-diaminopropionic acid,bismercaptoacetyldiaminosuccinic acid,N-(mercaptoacetyl-aminoethyl)cysteine dimercaptosuccinic acid,dimercapto-propionic acid, cysteine, cysteamine, diphosphinopropionicacid, and derivatives thereof wherein one or more thiol functions areprotected by a R₄ group, R₄ being selected from hydrogen and suitablethiol protective groups.
 11. The bis-indole derivative or a metalcomplex according to claim 1, having the structure shown in formula (Ia)hereunder

wherein L, C₁, C₂, R₁, R₂, R₃, p, q and r are as defined in claim
 1. 12.The bis-indole derivative or a metal complex according to claim 1, beingselected from the group consisting of:[{2-[N′-(3-{[2-N′-{2-[(2-{[2-bis(carboxymethylamino)-ethyl]carboxymethyl-amino}ethyl)carboxymethylamino]-acetyl}hydrazinocarbonyl)-1H-indol-3-yl]-phenyl-methyl}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-aceticacid,-[{2-[N′-(3-{[2-(N′-{2-[(2-{[2-bis(carboxymethyl-amino)-ethyl]carboxymethyl-amino]-ethyl)carboxymthyl-amino]-acetyl}-hydrazinocarbonyl)-1H-indole-3-yl]-methyl}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-{[2-bis(carboxymentyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-aceticacid,-{4,7-bis-carboxymethyl-10-[({3-[(2-{N′-[(4,7,10-tris-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetyl]-hydrazinocarbonyl}-1H-indol-3-yl)-methyl]-1H-indole-2-carbony}-hydrazino)-2-oxo-ethyl]-1,4,7,10-tetraaza-cyclododec-1-yl}-aceticacid, and-{4,8-bis-carboxymethyl-11-[({3-[(2-{N′-[(4,8,11-tris-caboxymethyl-1,4,8,11-tetraaza-cyclotetradec-1-yl)-acetyl]-hydrazinocarbonyl}-1H-indole-3-yl)-methyl]-1H-indol-2-carbonyl}-hydrazino)-2-oxo-ethyl]-1,4,8,11-tetraaza-cyclotetradec-1yl}-aceticacid, enantiomers and pharmaceutically acceptable salts thereof.
 13. Thebis-indole derivative or a metal complex according to claim 1, being thesodium salt of[{2-[N′-(3-{[2-(N′-{2-[(2-{[2-bis(carboxymethylamino)-ethyl]carboxymethyl-amino}ethyl)carboxymethyl-amino]-acetyl}hydrazinocarbonyl)-1H-indol-3-yl]-phenyl-methyl}-1H-indole-2-carbonyl)-hydrazino]-2-oxo-ethyl}-(2-55[2-bis(carboxymethyl-amino)-ethyl]-carboxymethylamino}-ethyl)-amino]-aceticacid.
 14. The bis-indole derivative or a metal complex according toclaim 1, being the sodium salt of{4,7-bis-carboxymethyl-10-[({3-[(2-{N′-[(4,7,10-tris-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetyl]-hydrazinocarbonyl}-1H-indol-3-yl)-methyl]-1H-indole-2-carbonyl}-hydrazino)-2-oxo-ethyl]-1,4,7,10-tetraaza-cyclododec-1-yl}aceticacid.
 15. the A bis-indole derivative or a metal complex according toclaim 1, the said metal being a radioactive metal selected from thegroup consisting of ^(99m)Tc, ¹¹¹In, ⁶⁷Ga, ⁹⁰Y, ¹⁸⁶Re and ¹⁸⁸Re.
 16. Thebis-indole derivative or a metal complex according to claim 1, the saidmetal being a non-radioactive metal selected from the group consistingof gadolinium, manganese and iron.
 17. A pharmaceutical compositioncomprising at least one pharmaceutically acceptable carrier and asubstituted bis-indole derivative of formula (I) or a metal complx,wherein said metal complex consisting of bis-indole dervatives offormula (I) and a radioactive or non-radioactive metal Ion of an elementwith an atomic number selected from the group consisting of 21 to 32, 37to 39, 42 to 44, 49, 50 or 57 to 83, and wherein the formula (I) ishereunder:

an enantiomer or a pharmaceutically acceptable salt thereof, or wherein:L represents a single bond or an optionally substituted linking agent(Li) which covalently links together the carbon atoms being respectivelyin positions 2 or 3 and 2′ or 3′ on the heterocyclic indole rings, (Li)being an optionally substituted alkylene group; R₁ and R₂ are optionalsubstituents of any free position of the phenyl rings of the indolylgroups and are each independently selected from halogen atoms, straightchain alkyl groups of 1 to 6 carbon atoms, branched chain alkyl groupsof 3 to 7 carbon atoms, cycloalkyl groups of 3 to 7 carbon atoms, alkoxygroups of 1 to 6 carbon atoms, nitro, trifluoromethyl, cyano, carboxylicacid, sulfonic acid, carboxylic acid esters wherein the ester groupderives from an alkyl group having 1 to 4 carbon atoms, substituted orunsubstituted carboxamides CO—NR₄R′₄ and substituted or unsubstitutedamines NR₄R′₄ wherein R₄ and R′₄ are each independently selected fromhydrogen and optionally substituted C₁–C₂₀ branched chain or straightchain alkyl groups or C₆–C₂₀ aryl groups or C₆–C₂₀ alkylaryl groups, R₃is an optional substituent of the linking agent (Li) and is selectedfrom optionally substituted aryl groups and optionally substitutedbranched chain or straight chain alkyl groups; q, p and r are integersindicating the number of the respective substituents R₁, R₂ and R₃ andare each independently selected from 0 to 4, provided that r is 0 when Lis a single bond; p1 C₁ and C₂ are metal chelating agent-containingsubstituents of the heterocyclic rings of the indolyl groups; m and nare integers indicating the number of the respective metal chelatingagent-containing substituents C₁ and C₂ and are each 0 or 1, providedthat the sum of m and n is at least 1; and wherein R₁, R₂ or R₃independently does not renresent a heterocycre or heteroaryl, andwherein R₁, R₂ or R₃ independently is not substituted with a heterocycleor heteroaryl.